BRPI0214840B1 - methods for detecting human oncogenic mutations, automated methods for detecting a mutation and method and assay for identifying one or more compounds having anti-proliferative activity - Google Patents

Abstract

"GENES". The invention relates to mutations in the B-Raf gene products. The described mutations are identified in human tumors of natural origin. These mutations are associated with cancer phenotypes and can be used as a basis for diagnosing cancer, cancer cells or a predisposition to cancer in human individuals, and for developing anti-cancer therapies.

Description

FIELD OF THE INVENTION

[001] The present invention relates to cancer-specific mutants of B-raf genes and their uses in the detection of abnormal cells and cancer. In addition, the invention describes methods for the diagnosis of cancer, the detection of cancer cells in individuals and the development of therapeutic agents for the treatment of cancer.

INTRODUCTION

[002] Cancer can develop in any tissue of any organ, at any age. Most cancers detected at a primary stage are potentially curable, so the ability to screen patients for early signs of cancer, and thus allow intervention early, is highly desirable (see, for example, the Merck Manual of Diagnosis and therapy (1992) 16th ed. Merck & Co).

[003] Cancer cells show unregulated growth, lack of differentiation and capacity to invade local tissues and evolve in metastasis. Thus, cancer cells are different from normal cells, and are potentially identifiable, but not only for their phenotypic traits, but also for their molecular biochemical and biological characteristics. Such characteristics, in turn, are dictated by changes in cancer cells, which occur at the genetic level, in a subset of cellular genes known as oncogenes, which directly or indirectly control cell growth and differentiation.

[004] The Raf oncogene family includes three highly conserved genes called A-, B- and C-raf (also called raf-1). C-Raf, the most well-characterized element of the raf family, is the cellular homologue of v-raf the 3611 murine sarcoma virus transforming gene. The viral raf oncogene encodes a protein that lacks the amino-terminal sequences of the normal Raf protein. These amino-terminal sequences are crucial for the regulation of RAF serine / threonine protein kinase activity, and their deletion or substitution results in the constitutive activity of the oncogene-encoded RAF protein. This unregulated activity promotes cell proliferation, resulting in cell transformation. The DNA of a few tumors was claimed to contain a transforming activity detectable by DNA transfection from NIH / 3T3 cells, identified as derived from truncated C-raf-1. However, these results are likely to be transfection artifacts, because the same mutations were not found in tumors from which the transforming DNA was derived. Mutations created artificially in the C-raf gene when introduced into cells in vitro can induce transformation.

[005] The B-raf gene is the human homologue of the poultry c-Rmil proto-oncogene encoding a 94 kD serine / threonine kinase detected in bird cells. This protein contains amino-terminal sequences not found in other proteins in the mil / raf gene family. These sequences are encoded by 3 exons in the poultry genome. Eychene et al. (1992) Oncogene 7: 1657-1660 reports that these 3 exons are conserved in the human B-raf gene and that they encode an amino acid sequence similar to that of the poultry gene. They identified 2 human B-raf sites: B-raf 1 which was mapped to 7q34 by fluorescence in situ hybridization and show to code the functional gene product and B-raf 2 an inactive processed pseudogene located at Xq13.

[006] By examining a mouse cDNA library with a v-raf oncogenetic probe, Huebner et al., (1986) Proc. Nat. Acad. Sci. 83: 3934-3938 isolated a transforming raf-related cDNA, A-raf that represented a distinct raf1 gene. The mouse's only A-raf site and the man's A-raf1 site are actively transcribed in several mouse and human cell lines. The complete 606 amino acid sequence of the human A-raf1 oncogene was deduced from the 2,453 nucleotide sequence of the cDNA. The A-raf gene is linked to X.

[007] A known mechanism for converting proto-oncogenes to oncogenes is the appearance of single mutations in the DNA sequence, known as point mutations, which results in a change in the amino acid sequence of the encoded polypeptide. For example, ras oncogenes are not present in normal cells, but their proto-oncogen counterparts are present in all cells. Proteins Wild-type frogs are small GTP-binding proteins that are involved in signal transduction. However, many ras oncogenes of viruses and human tumors have a point mutation in codon number 12: the codon GGC that normally encodes glycine is changed to GTC, which encodes valine. Multiple mutations have been documented in this codon, including at least 5 different substitutions that are activation. This single amino acid change prevents the GTPase activity of the Ras protein, and makes Ras essentially activated, as it remains bound to GTP. The amino acids at positions 13 and 61 are also frequently raised in the ras oncogenes of human tumors. Raf protein is a serine / threonine kinase that is structurally related to the family (protein kinase C PKC, and is essential in cell growth and differentiation. Raf proteins are involved in signal transduction in MAP kinase activation which is highly conserved in eukaryotic organisms, MAP kinases (mitogen-activated protein kinases), which include ERK1 and ERK2, directly phosphorylated transcription factors to regulate biological events, MAPKKs (MAP kinase kinases) and MAPKKKs (MAPKK kinases) in turn, regulate MAP kinases.

[008] Raf proteins are MAPKKKs and are believed to phosphorylate MAPKKK MEK in vivo in mammalian biological systems. Distinct raf genes encode A-Raf, B-Raf, and Raf-1 (also known as c-Raf) in vertebrates (reviewed in Papin et al., 1998, Oncogene 12: 2218-2221). The three proteins are not equal in their ability to activate MEK. A-Raf, the less well-characterized member of the family, appears to be a weak MEK activator, its activity being difficult to measure (Pritchard et al., 1995, Mol. Cell. Biol. 15, 6430-6442). B-Raf and Raf-1 also differ in their ability to activate MEK. Although Raf-1 is omnipresent, B-Raf demonstrates the highest levels of expression in neural tissues (Barnier et al., 1995, J. Biol. Chem. 270, 23381-23389). However, B-RAf has been identified as the main MeK activator, even in cells where its expression is rarely detectable by western staining analysis (Catling et al., 1994, Jaiswal et al., 1994; Reuter et al., 1995 ; Huser et al, 2001, Mikula et al., 2001). With persistence, B-Raf has a greater affinity for MEK-1 and MEK-2 than Raf-1 (Papin et al., 2996, Papin et al. 1998) and is more efficient in MAPKK MEK phosphorylation.

[009] The activator upstream of B-Raf is GTPase Frogs. A number of Ras isoforms exist in mammals, N-Ras, Ha-Ras, Ki-Ras4A and Ki-Ras4B. Other GTPases of the RAS superfamily can also interact with B-Raf. For example, Rap-1, reviewed in Peysonnaux et al., (2001) Biology of the Cell 93: 53-62 appears to be a selective B-Raf activator.

SUMMARY OF THE INVENTION

[0010] Spot mutants in B-Raf gene products are described herein. The described point mutations are identified in human tumors of natural origin. These dot mutations are associated with cancer phenotypes and can be used as a basis for diagnosing cancer, cancer cells or a predisposition to cancer in human individuals.

[0011] Since many signaling pathways that are mediated by the activation of B-Raf kinase activity are involved in the control of cell proliferation and oncogenetic transformation, it would be convenient to be able to quickly detect changes in the B-raf gene which can result in a oncogenetic character.

[0012] Thus, in a first aspect, a naturally occurring cancer-associated mutant of a human B-RAf polypeptide comprising one or more mutations is provided.

[0013] Preferably, the cancer-associated mutant is isolated from a naturally occurring primary human tumor.

[0014] Preferably, the mutation is located in the B-RAf kinase domain.

[0015] The present invention provides several of such mutations, which have been seen to be associated with a cancerous phenotype in human cancers, and thus, establish a link between B-RAf mutations and cancer in vivo.

[0016] Preferably, the mutation is a point mutation. Mutations can also include changes such as insertions, deletions, or substitutions of one or more nucleotides, preferably 2, 3, 4, 5 or 6 nucleotides.

[0017] Advantageously, the mutations are located at the C-terminal position to amino acid 300 in B-Raf. Preferred positions are 463, 465, 468, 585, 594, 595, 596 and 599.

[0018] In a more preferred embodiment, mutations are selected from the group consisting of V599E, V599D, G595R, G465V, G465E, G465A, G468A, G468E, E585K, F594L, G595R, L596V, L596R and G463E.

[0019] Preferably the polypeptide is isolated.

[0020] The invention furthermore encompasses fragments of the polypeptides according to the invention where such fragments include the mutation as described.

[0021] In a second aspect, a nucleic acid encoding a mutant B-Raf polypeptide or fragment thereof according to the present invention is provided. Preferably the nucleic acid comprises one or more point mutations.

[0022] Preferably the nucleic acid is isolated.

[0023] Spot mutations in B-raf genes have been detected showing association with tumors. Advantageously, the point mutation occurs at one or more of B-raf positions 1388, 1394, 1403, 1753, 1782, 1783, 1796, 1797, 1787 and 1786. Preferably the point mutation is G1388T, G1783C, TG1796-97AT, G1394T, G1394A, G1394C, G1403C, G1403A, G1753A, T1782G, G1388A, T1796A, 61787G or C1786G in B-raf.

[0024] The invention furthermore provides the complement of any nucleic acid described above.

[0025] In a further embodiment, a nucleic acid is provided which specifically hybridizes to a nucleic acid according to the invention as written herein. Such a nucleic acid can for example be a primer that directs the specific amplification of a nucleic acid encoding mutant B-Raf according to the invention in a nucleic acid amplification reaction.

[0026] In a third aspect, the invention provides a linker that selectively binds to a mutant B-Raf polypeptide according to the invention.

[0027] Such a linker is advantageously an immunoglobulin, and is preferably an antibody or an antigen-binding fragment thereof.

[0028] In accordance with a fourth aspect, a method for detecting cell transformation is provided, comprising the steps of: (a) isolating a sample of cellular material from an individual, (b) examining the nucleic acid material of hair at least a part of one or more B-raf genes in said cell material, and (c) determining whether such a nucleic acid material comprises one or more mutations in a sequence encoding a B-Raf polypeptide.

[0029] Advantageously the mutation is a point mutation.

[0030] Advantageously the mutation occurs in one or more of positions 1388, 1394, 1403, 1753, 1783, 1783, 1796, 1797, 1787 and 1786 and B-raf. Preferably the point mutation is G1388T, G1783C, 6G1796-97AT, G1394T, G1394A, G1394C, G1403C, G1403A, G1753A, T1782G, G1388A, T1796A, 61787G or C1786G in B-raf.

[0031] The mutations identified according to the invention are, advantageously, somatic mutations, which occurred in somatic tissue and are not transmitted through the germline. Thus, the invention also relates to a method for detecting transformation, comprising the steps of: (a) isolating a first sample of cellular tissue material from an individual, suspected of having cancer, and a second sample of the material of a non-cancerous tissue from the same individual, (b) examining the nucleic acid material from at least part of one or more B-raf genes in both samples of cellular material, and (c) determining whether such a Nucleic acid comprises one or more mutations per point in a sequence encoding a B-Raf polypeptide, and such a mutation being present in the cellular material of the tissue suspected of cancer, but not present in the cellular material of the non-cancerous tissue.

[0032] The invention furthermore provides a method for detecting cell transformation, comprising the steps of: (a) obtaining a sample of cellular material from an individual, (b) examining such sample with a ligand that selectively binds to a polypeptide Mutant B-Raf according to the invention, and (c) detecting one or more mutant B-Raf polypeptide in said sample.

[0033] In a still further aspect, the invention relates to a method for identifying one or more compounds with anti-proliferative activity, comprising the following steps: (a) providing one or more mutant B-Raf polypeptide according to the present invention, (b) contacting such polypeptide (s) with one or more compounds to be tested and (c) detecting an interaction between one or more compounds and such mutant polypeptides.

[0034] Preferably, the interaction is a connection interaction.

[0035] In addition, the invention provides an assay to identify one or more compounds with anti-proliferative activity, comprising the steps of: (a) providing one or more mutant B-Raf polypeptides according to the invention, (b) providing a substrate downstream for the B-Raf polypeptide, (c) detecting modification of the substrate in the presence of the test compound (s).

[0036] B-RAf is a protein kinase, and therefore, its substrates are capable of phosphorylated or dephosphorylated beings. Preferably the action of the B-Raf mutant on the substrate results in an identifiable change there. Advantageously, the substrate is an additional kinase or phosphatase that, in turn, modifies a third molecule in which a detectable change occurs.

[0037] For example, the substrate may be the MEK kinase. Phosphorylation of MEK can be detected directly, or preferably, detected by activating MEK to phosphorylate MAP Kinase.

[0038] Advantageously, a reference activity of the B-Raf mutant in the substrate is established and the activity in the presence and / or absence of the test compound (s) compared with the reference value. A reduction in the activity of the B-Raf mutant is indicative of a reduction in proliferative activity.

[0039] The invention furthermore provides a cell-based assay for selecting compounds with anti-proliferative activity. In a first embodiment, the invention provides a 3T3 focus formation assay comprising the steps of: (a) providing a culture of NIH 3T3 cells (b) transfecting such cells with a mutant B-raf nucleic acid according to the invention, ( c) exposing such cells to one or more test compound (s), and (d) determining the difference in the number of foci formed between the transfected cells exposed to such compound (s) for this and transfected cells without such exposure.

[0040] The cell-based assay is normally performed using 3T3 nIH cells. However, other cell types, especially fibroblasts, can be used in such an assay.

[0041] Advantageously, a reference formation activity of a mutant B-raf gene in the cells used in the assay is established, and the activity in the presence and / or absence of the compound (s) for the test compared with the reference value. A decrease in the focus-forming activity of the mutant B-raf gene is indicative of a reduction in proliferative activity, and therefore, anti-proliferative activity in the test compound (s).

[0042] Automated methods and apparatus for detecting mutations according to the invention are also provided.

BRIEF DESCRIPTION OF THE FIGURES

[0043] Figure 1A: B-Raf activity tests. B-Raf kinase activity was measured in a cascade assay by kinase immunoprecipitation using MBP as the final substrate. The activity is shown as the number of counts incorporated into the MBP. The test was performed in triplicate and the mean is shown, with error bars to represent deviations from the mean. Both the basal kinase activity (empty bars) and the kinase activity stimulated by RasV12 (hatched bars) are shown.

[0044] Figure 1B: B-Raf activity tests. B-Raf kinase activity was measured in a cascade kinase immunoprecipitation assay using MBP as the final substrate. The activity is shown as the number of points incorporated into the MBP. The test was performed in triplicate and the mean is shown, with error bars representing deviations from the mean. Both basal kinase activity (empty bars) and RasV12-stimulated kinase activity are shown.

[0045] Figure 2: Transformation of NIH3T3 cells by B-Raf and mutants and activation. The cells were transfected with the indicated constructors and the number of colonies was determined. The results are the average of at least three trials. The number of colonies relative to the number induced by B-Raf is shown.

[0046] Figure 3A. V599D is an activating mutation in BRAF. BRAF or BRAFV599D were expressed alone, or together with oncogenic RAS, as indicated. BRAF protein activity was determined using a cascade kinase immunoprecipitation assay in which the immunoprecipitated BRAF is used to sequentially activate MEK and ERK. ERK activation is determined using myelin basic protein and ATP [P32] -y as substrates.

[0047] Figure 3B. Inhibition of ERK in melanoma cell lines using pharmacological reagents. WM266.4 or A375P cells were treated with 10pM U0126, 10pM BAY 43-9006 or DMSO as a control. Equivalent amounts of cellular proteins were resolved in SDS gels and active ERK levels were determined using the ppERK antibody.

[0048] Figure 4. Inhibition of cell growth by pharmacological agents. WM-266.4 cells were incubated in the presence of U0126 (10 pM) or BAY 43-9006 (10 pM) or vehicle control (DMSO. After 48 hours, DNA synthesis was determined by incubating the cells with thymidine [H3] and the levels of thymidine incorporated into the cellular DNA were determined.

[0049] Figure 5A. CRAF expression is suppressed by siRNA. WM-266.4, Colo 829 or BE cells were treated with a CRAF-specific siRNA probe (CRAF), the disordered siRNA genetic probe, oligofectamine (oligo) or untreated (control). The cells were incubated for 24 hours and CRAF protein levels were determined by Western blotting.

[0050] Figure 5B. The BRAF expression is suppressed by siRNA. WM-266.4, Colo 829 or BE cells were treated with a BRAF specific siRNA probe, the disordered siRNA probe or were left untreated as shown. The cells were incubated for 24 hours and the BRAF activity levels were tested using a MEK and ERK kinase immunoprecipitation assay as sequential assays. ERK activity was determined using MBP and ATP [P32] -y as substrates.

[0051] Figure 6. BRAF ablation, but not CRAF blocks ERK activity in melanoma cells. WM-266.4 or Colo 829 cells were treated with a BRAF-specific sirNA probe (BRAF), or competition control (sBRAF) or a CRAF-specific probe (CRAF) or its competitive control (sCRAF), or oligophectamine (oligo) or left untreated) control) as indicated. The cells were incubated for the indicated times, and Colo 829 cells were treated for 24 hours. The levels of ERK activity in equivalent amounts of cell extract were determined by Western blotting with the ppERK antibody.

[0052] Figure 7. BRAF ablation, but not CRAF induces apoptosis in melanoma cells. WM-266.4 cells were treated with a BRAF specific siRNA probe (BRAF) or competitive control (sBRAF) or a CRAF specific siRNA probe (CRAF), or U0126 or DMSO 9oligo) or left untreated (control) as indicated. The cells were incubated for 96 hours and the cell cycle profile was analyzed by FACS or the expression PARP was examined by Western staining.

[0053] Figure 8. The validity of B-Raf and GST - MKKI activity. The assay performed using WTS 1B-RAF lysate (batch A), GST MKKI (6.5 pg / ml) and ERK2 (competent kinase, 100 pg / ml) to measure incorporation of P33-y-phosphate into MBP (0.3 mg / ml). The data shown are the mean ± SD of triplicate determinations.

[0054] Figure 9. Evaluation of the FlahsPlate Filter Plate and Radiometric Test Platform. The assay was performed using B-RAf1 WTS1 lysate (batch A) and GST MKKL (6.5 pg / ml) to measure incorporation of P33-y-phosphate into GST-kdERK2 (100 pg / ml). The data shown are the mean ± SD of determinations and triplicate.

[0055] Figure 10. Evaluation of DELFIA non-radiometric test platform. 100 ng GST-kdERK2 was pre-attached to each reservoir followed by addition of B-RAf lysate (batch A), GST-MKKL (6.5 pg / ml and ATP (500pM). The data shown are the mean ± SD of triplicate determinations.

[0056] Figure 11. Titration of anti-fluffy-ERK2 in DELFIA Assay. 100 ng GST-kdERK2 was pre-attached to the reservoir followed by addition of B-RAf lysate (lot A), GST-MKKL (6.5 pg / ml) and ATP (500 pM). The data shown is the average of duplicate determinations.

[0057] Figure 12. Titration of secondary antibody labeled with europium in DELFIA Assay. 100 ng of GST-kdERK2 was pre-attached to the reservoir followed by the addition of B-RAf lysate (batch A), GST-MKKL (6.5 pg / ml) and ATP (500 pM) The data show the mean of determinations in duplicate.

[0058] Figure 13. Evaluation of Homogeneous Test Protocol. The homogeneous assay was performed on a 96-well plate using a reaction volume of 50 pl containing a B-RAf lysate (batch B), 6.5 pg / ml GST-MKKL, 80 pg / ml GST-kdERK2 and 500 pM ATP. The data shown are the mean ± SD of triplicate determinations.

DETAILED DESCRIPTION OF THE INVENTION

[0059] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning normally understood by those skilled in the art (for example, in cell culture, molecular genetics, nucleic acid chemistry, hybridization and bio techniques -chemistry). Standard techniques are used for molecular, genetic and biochemical methods. See, generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 2. ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4a. Ed., John Wiley & Sons, Inc., as well as Guthrie et al., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol, 194, Academic Press, Inc .., (1991), PCR Protocols: A Guide to Methods and Applications (Innis, et al., 1990. Academic Press, San Diego, Calif.), McPherson et al., PCR Volume 1, Oxford University Press, (1991), Culture of Animal Cells: A Manual of Basic Technique , 2a. Ed (R. I. Freshney. 1987. Liss, Inc. New York, N.Y), and Gene Transfer and Expression Protocols, p. 109-128 ed. E. J. Murray, The Human Press Inc. (Clifton, N.J). These documents are hereby incorporated by reference.

Definitions:

[0060] The present application describes mutants of B-Raf polypeptide. As used herein, the term "RAF Polypeptide" is used to denote a polypeptide from the RAF family. RAF was first identified in a type C virus, a replication defective, critically transformed, singularly cloned, which contained a v-raf oncogene (Rapp, et al., Proc. Nat. Acad. Sci 80: 4218-4222. 1983) . The cellular homologue, c-raf, is present in mammalian DNA. Other counterparts have since been discovered in humans and birds, where raf was seen as the counterpart of the thousand bird oncogene. B-RAf is related to RAF but has three additional N-terminal exons. The term "B-Raf" thus encompasses all known human B-RAf homologues and variants, as well as other polypeptides, which show sufficient homology to B-RAf for identification as B-RAf homologs. The terms do not include ARAF, CRAF or RAF1. Preferably, B-Raf, is identified as a polypeptide having the sequence shown in accession number NP_004324, nucleic acid accession number NM_004333.

[0061] The term "B-RAf" preferably includes polypeptides that are 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to NP_004324. Homology comparisons can be made by estimate, or more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate perceptual homology (%) between two or more strings.

[0062] Percentage homology can be calculated during adjacent sequences, that is, one sequence is aligned with the other sequence and each amino acid in a sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called a “no gap” alignment. Typically, such gap-free alignments are performed only on a relatively small number of residues (for example, less than 50 adjacent amino acids).

[0063] Although this is a very simple and concise method, it fails to take into account the fact that, for example, in a pair of sequences otherwise identical, an insertion or deletion will cause the following amino acid residues to be placed out of alignment, potentially resulting in a large reduction in percentage homology when global alignment is performed. Consequently, most of the sequence comparison methods are designed to produce optimal alignments, which take into account possible insertions and deletions, without unduly penalizing the global homology mark. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.

[0064] However, these more complex methods confer “gap disadvantages” for each gap that occurs in alignment, so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting greater runs -lation between two compared strings - you will get more marks than one with many intervals. "Related interval costs" are typically employed, which are relatively more expensive due to the existence of an interval and a smaller disadvantage for each subsequent residue in the interval. This consists of the most commonly used range marking system. Disadvantages with large intervals will obviously produce optimized alignments with smaller intervals. Most alignment programs allow you to modify the alignment disadvantages. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Besfit package (see below) the disadvantage in the default range for amino acid sequences is -12 for a range and -4 for each extension.

[0065] The calculation of the maximum percentage homology, therefore, first requires the production of an optimal alignment, taking into account interval disadvantages. A suitable computer program to perform such an alignment is the GCG Wisconsin Besfit package (University of Wisconsin U.S.A. Devereux et al., 1984 Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - chapter 18), GASTA (Atschul et al, 1990, J. Mol. Biol. 403 -410) and the GENEWORKS set of comparison tools. Both BLAST and FASTA are available for offline and online research (see Ausubel et al., 1999 ibid pages 758 to 7-60). However, it is preferred to use the GCG Bestfit program.

[0066] Although the final percentage homology can be measured in terms of identity, the alignment process itself is not typically based on an "all or nothing" pair comparison. In contrast, a staggered similarity marking matrix is generally used that determines markings for each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. Wisconsin GCG programs generally use either the public default values or a standard symbology comparison table if provided (see User Manual for details). It is preferred to use public default values for the GCG package, or in the case of other software, the default matrix such as BLOSUM62.

[0067] Once the software has produced an optimal alignment, it is possible to calculate the percentage of homology, preferably percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0068] A "fragment" of a polypeptide according to the invention is a fragment of polypeptide that comprises the mutant amino acids described according to the invention. The fragment can be of any length up to the total length of the B-Raf polypeptide, it thus encompasses the B-Raf polypeptides that have been truncated by a few amino acids, as well as shorter fragments. Advantageously, the fragments are between about 764 and about 5 amino acids in length, preferably about 5 to about 20 amino acids in length, advantageously between about 10 and about 50 amino acids in length. The fragments according to the invention are useful, inter alia, in immunizing animals to create antibodies. Thus, the polypeptide fragments according to the invention advantageously comprise at least one antigenic determinant (epitope) characteristic of the B-RAf mutant as described herein. Wherever a particular polypeptide fragment has such antigenic properties, it can be readily determined by routine methods known in the art. Peptides composed of at least six amino acid residues are often known to elicit an immune response.

[0069] A "nucleic acid" of the present invention is a nucleic acid that encodes a human B-Raf polypeptide as above. The term, moreover, includes polynucleotides capable of hybridizing, under stringent hybridization conditions, to naturally occurring nucleic acids, as identified above, or in their supplements. “Strict hybridization conditions” refers to an overnight incubation at 42 ° C in a solution comprising 50% formamide, 5x SSC (750 nM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6) solution 5x Denhardt, 10% dextran sulfate, and 20 pg / ml of scraped, denatured salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65 ° C.

[0070] Although nucleic acids, as referred to herein, are generally naturally occurring nucleic acids, the term may include in its scope, modified, artificial nucleic acids, having modified central structures, or bases, as is known in the art.

[0071] A nucleic acid encoding a fragment according to the invention can be the result of the amplification of nucleic acid from a specific region of a B-raf gene, incorporating a mutation according to the present invention.

[0072] A polypeptide or nucleic acid "isolated" as used here, refers to material removed from its original environment (for example, the natural environment in which it occurs in nature) and is thus altered by the hand of the man of his state Natural. For example, an isolated polynucleotide can be part of a vector or composition of matter, or it can be contained in a cell and still be isolated ”because that particular vector, composition or matter, or cell is not the original medium of the polynucleotide. Preferably, the term "isolated" does not refer to genomic or cDNA libraries, complete whole cell or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred over blots), preparations of whole cell genomic DNA that have been scattered or other compositions where the technique does not demonstrate distinctive characteristics of the polypeptides / nucleic acids of the present invention.

[0073] The polypeptides according to the invention comprise one or more mutations.

[0074] "Mutations" include addition, deletion or substitution of amino acids, advantageously it refers to substitutions of amino acids. Such mutations at the polypeptide level are reflected at the nucleic acid level by addition, deletion or substitution of one or more nucleotides. Generally, such mutations do not alter the nucleic acid reading. Advantageously, changes at the nucleic acid level are point mutations, in which a single nucleotide is replaced by another, changing the codon of which it is a part to specify a different amino acid.

[0075] The B-Raf mutations identified in the present invention occur naturally, and were not intentionally induced in cells or tissue by the application of carcinogens or other tumorigenic factors. Thus, the mutations identified here accurately reflect natural tumorigenesis in human tissues in vivo. Its detection is thus a much better basis for diagnosis than the detection of mutations identified in rodents after artificial chemical tumor induction.

[0076] A "somatic" mutation is a mutation that is not transmitted through the germination line of an organism, and occurs in somatic tissues of the organism. Advantageously, a somatic mutation is one that is determined to be somatic, through analysis of the normal sample / tumor sample.

[0077] All the amino acid and nucleotide numbers used here start from the +1 amino acid of the B-Raf polypeptide or from the first ATG of the nucleotide sequence that encodes it.

[0078] "Amplification" reactions are nucleic acid reactions that result in specific amplification of target nucleic acids over non-target nucleic acids. The polymerase chain reaction (PCR) is a well-known amplification reaction.

[0079] An "immunoglobulin" belongs to a family of polypeptides that has the immunoglobulin fold characteristic of immunoglobulin molecules (antibody), which contains two p layers and usually a conserved disulfide bridge. Members of the immunoglobulin superfamily are involved in many aspects of cellular and non-cellular interactions in vivo, including functions throughout the immune system (eg, antibodies, T cell receptor molecules and the like), involvement in cell adhesion (eg, the IÇAM molecules) and intracellular signaling (for example, receptor molecules such as the PDGF receptor). The present invention is preferably applicable to antibodies, which are able to bind to target antigens with high specificity.

[0080] "Antibodies" can be complete antibodies, or fragments of these that bind to the antigen. For example, the invention includes fragments such as Fv and FAB, as well as Fab'e F (ab ') 2, and antibody variants such as scFv, single domain antibodies, DAb antibodies and other molecules based on the antigen binding antibody.

[0081] "Cancer" is used here to refer to the neoplastic growth that arises from the cellular transformation into a neoplastic phenotype. such cellular transformation often involves genetic mutation. in the context of the present invention, transformation involves genetic mutation by altering one or more B-raf genes as described herein.

Methods for Detection of Nucleic Acids

[0082] Detection of mutant nucleic acids encoding B-Raf can be employed in the context of the present invention to diagnose the presence or predisposition to cell transformation and cancer. Since mutations in B-raf genes generally occur at the DNA level, the methods of the invention can be based on detecting mutations in genomic DNA, as well as in the transcripts and proteins themselves. It may be convenient to confirm mutations in genomic DNA by analyzing transcripts and / or polypeptides to ensure that the detected mutation is, in fact, expressed in the individual.

[0083] Mutations in the genomic nucleic acid are advantageously detected by techniques based on the deviation of mobility in enlarged nucleic acid fragments. For example, Chemn et al., Anal. Biochem 1996 Jul. 15; 239 (1): 61-9, describe the detection of mutations with a single basis by a competitive mobility shift assay. In addition, assays based on the technique of Marcelino et al., Bio Techniques 26 (6): 1134-1148 (June 1999) are commercially available.

[0084] In a preferred example, different capillary duplex analysis can be used to detect the presence of mutations based on the shift in duplex nucleic acid mobility in capillary systems as a result of the presence of incompatibilities.

[0085] The generation of nucleic acids for analysis of samples generally requires amplification of the nucleic acid. Many amplification methods rely on the enzyme chain reaction (such as a polymerase chain reaction, a ligase chain reaction or a self-maintained sequence replication) or the replication of all or part of the vector to which it has been cloned . Preferably, the amplification according to the invention is an exponential amplification as shown, in, for example, polymerase chain reaction.

[0086] Many methods of target and signal amplification have been described in the literature, for example, general consultations of these methods in Landegren, U., et al., Science 242: 229-237 (1988) and Lewis, R., Genetic Engineering News 10: 1, 54-55 (1990). These amplification methods can be used in the methods of the present invention, and include polymerase chain reaction (PCR), in situ PCR, ligase amplification reaction (LAR), ligase hybridization, bacteriophage Qbeta replicase, amplification system with transcription based (TAS), genomic amplification with transcript sequencing (GAWTS), amplification based on the nucleic acid sequence (NASBA) and in situ hybridization. Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.

Polymerase Chain Reaction (PCR)

[0087] PCR is a nucleic acid amplification method described inter alia, in U.S. Patent Nos. 4,683,195 and 4,683,202. PCR consists of repeated DNA polymerase cycles of extension reactions of the primer generated by DNA polymerase. The target DNA is denatured with heat and two oligonucleotides, which match the target sequence in opposite strands of the DNA to be amplified, are hybridized. These oligonucleotides become primers for use with DNA polymerase. The DNA is copied by extending the primer to make a second copy of both strands. Repeating the thermal denaturation cycle, the hybridization of the primer and extension of the target DNA can be amplified one million times or more in about two to four hours. PCR is a molecular biology tool, which must be used in conjunction with a detection technique to determine the results of the amplification. An advantage of PCR is that it increases sensitivity by amplifying the amount of target DNA by 1 million to 1 billion times in approximately 4 hours. PCR can be used to amplify any known nucleic acid in a diagnostic context (Mok et al., (1994), Gynaecologic Oncology, 52: 247-252).

Independent Sequence Replication (3SR)

[0088] Independent sequence replication (3SR) is a variation of the TAS sequence, which involves the isothermal amplification of a nucleic acid matrix via sequential reverse transcriptase (RT) turns, polymerase and nuclease activities that are mediated by an enzymatic cocktail and appropriate oligonucleotide primers (Guatelli et al. (1990) Proc. Natl. Acad. Sci USA 87: 1874). The enzymatic degradation of the RNA of the RNA / DNA heteroduplex is used in place of thermal denaturation. RNAse H and other enzymes are added to the reaction and all steps take place at the same temperature and if more reagent additions. Following this process, amplifications from 106 to 109 were achieved in one hour at 42 ° C.

Link Amplification (LAR / LAS)

[0089] Link amplification reaction or link amplification systems uses DNA ligase and four oligonucleotides, two for each target strand. This technique is described by Wu, D. Y and Wallace, R.B (1989) Genomics 4: 560. The oligonucleotides hybridize to adjacent sequences in the target DNA and are joined by ligase. The reaction is thermo-denatured and the cycle repeated.

QB Replicase

[0090] In this technique, RNA replicase for bacteriophage QB, which replicates single-stranded RNA is used to amplify the target DNA, as described by Lizardi et al., (1988) Bio / Technology 6: 1197. First, the target DNA is hybridized to a primer including a T7 promoter and a 5 'QB sequence region. Using this primer, reverse transcriptase generates a cDNA that connects the primer to its 5 'end in the process. These two steps are similar to the TAS protocol. The resulting heteroduplex is thermo-denatured. Next, a second primer containing a region of the QB 3 'sequence is used to start a second round of cDNA synthesis. This results in a double-stranded DNA containing both the 5 'and 3' ends of the bacteriophage QB, as well as a T7 RNA polymerase binding site. T7 RNA polymerase then transcribes the double-stranded DNA into its new RNA, which mimics QB. After extensive washing to remove any unhybridized probe, the new RNA is eluted from the target and replicated by QB replicase. The last reaction creates amplification increased by 107 in about 20 minutes.

[0091] Alternative amplification technology can be explored in the present invention. For example, current circle amplification (Lizardi et al., (1998) Nat Genet 19: 225) is a commercially available amplification technology (RCATtm) that is powered by DNA polymerase and can replicate circular oligonucleotide probes with linear or linear kinetics. geometry under isothermal conditions.

[0092] In the presence of two suitably designed primers, there is a geometric amplification via displacement of DNA strand and hyper-branching to generate 1012 or more copies of each circle in 1 hour.

[0093] If a single primer is used, RCAT generates in a few minutes, a linear chain of thousands of copies of DNA linked in tandem from a target covalently linked to that target.

[0094] Another technique, amplification by displacement of the filament (SDA; Walker et al., (1992) PNAS (USA) 80: 392) starts with a specially defined sequence unique to a specific target. But unlike other techniques that rely on thermal cycling, SDA is an isothermal process that uses a series of primers, DNA polymerase and a restriction enzyme to exponentially amplify the unique nucleic acid sequence

[0095] SDA comprises both a target generation phase and an exponential amplification phase.

[0096] In a target generation, double-stranded DNA is thermally denatured, creating two single-stranded copies. A series of specially manufactured primers combine with DNA polymerase (amplification primers for copying the base sequence and bumper primers for displacing newly created strands) to form altered targets capable of exponential amplification.

[0097] An amplification primer is attached to each strand its complementary DNA sequence. DNA polymerase then uses the primer to identify a site to extend the primer from its 3 'end, using the altered target as a template for adding individual nucleotides. The extended primer thus forms a double-stranded DNA segment containing a complete restriction enzyme recognition site at each end.

[0098] A restriction enzyme is then linked to the double-stranded DNA segment at its recognition site. The restriction enzyme dissociates from the recognition site after having only cleaved a filament from the two-sided segment, forming a cut. DNA polymerase recognizes the cut and extends the filament from the site, displacing the previously created filament. The recognition site is thus repeatedly cut and restored by the restriction enzyme and DNA polymerase with continuous displacement of the DNA strands containing the target segment.

[0099] Each displaced filament is then available for annealing with amplification primers as above. The process continues with repeated perforation, extension and displacement of the new DNA strands resulting in an exponential amplification of the original target DNA.

[00100] Once the nucleic acid has been amplified, a number of techniques are available for detecting single base pair mutations. One such technique is Single Stranded Conformational Polymorphism (SSCP). Detection of SCCP is based on aberrant DNA migration that has undergone a single strand mutation compared with the reference DNA during electrophoresis. The mutation produces conformational alteration in a single single-stranded DNA, resulting in the deviation of mobility. Fluorescent SCCP uses fluorescently labeled primers to aid in detection. Reference and mutant DNA are thus amplified using fluorescently labeled primers. The amplified DNA is denatured and suddenly cooled to produce single-stranded DNA molecules, which are examined by un-denatured gel electrophoresis.

[00101] Inadequate chemical cleavage (CMC) is based on the recognition and cleavage of mismatched base pairs of DNA by a combination of hydroxylamine, osmium tetroxide and piperidine. Thus, both reference DNA and mutant DNA are amplified with fluorescent labeled primers. Amplicons are hybridized and then subjected to cleavage using osmium tetroxide, which binds to a poorly combined T base or hydroxylamine, which binds to poorly combined C base, followed by piperidine that cleaves at the site of a modified base. Divided fragments are then detected by electrophoresis.

[00102] Techniques based on restriction fragment polymorphisms (RFLPs) can also be used. Although many simple nucleotide polymorphisms (SNPs) do not allow conventional RFLP analysis, primer-induced restriction analysis PCR (PIRA-PCR) can be used to introduce restriction sites using PcR primers in an SNP-dependent mode. Primers for PIRA-PCR that introduce suitable restriction sites can be designed by computational analysis for example, as described in Xiaiyi et al, (2001) Bioinformatics 17: 838-839.

[00103] In an alternative embodiment, the present invention provides the detection of gene expression at the level of RNA. Typical test formats using ribonucleic acid hybridization include nuclear composition assays, PCR-RT and RNase protection assays Melton et al., Nucleotide. Acids Res. 12: 7035. Detection methods that can be employed include radioactive labels, enzyme labels, chemiluminescent labels, fluorescent labels and other suitable labels.

[00104] PCR-RT is used to amplify RNA targets. In this process, the enzyme reverse transcriptase is used to convert RNA into complementary DNA (cDNA), which can then be extended using PCR. This method has proven to be useful for the detection of viral RNA. Its application is, in other aspects with regard to PCR, described above.

Methods for Detecting Polypeptides

[00105] The invention provides a method where a protein encoded by a mutant B-raf gene is detected. Proteins can be detected by protein gel assay, antibody binding assay or other detection methods known in the art.

[00106] For example, therefore, mutant B-RAf polypeptides, can be detected by differential mobility in protein gels, or by other size analysis techniques such as mass spectrometry, in which the presence of the mutant amino acids can be determined from according to molecular weight. Peptides derived from mutant B-Raf polypeptide in particular are susceptible to differentiation by size analysis

[00107] Advantageously, the detection means are sequence specific, such that a particle point mutation can be identified precisely in the mutant B-RAf polypeptide. For example, polypeptide or RNA molecules can develop, specifically recognizing mutant B-Raf polypeptide in vivo or in vitro.

[00108] For example RNA aptamers can be produced by SELEX. SELEX is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules. It is described, for example, in U.S. patents 5654151, 5503978, 5567588 and 5270163, as well as in PCT publication WO 96/38579, each of which is now specifically incorporated by reference.

[00109] The SELEX method involves the selection of nucleic acid aptamers, single-stranded nucleic acids capable of binding to a desired target from an oligonucleotide library. Starting from a library of nucleic acids, preferably comprising a segment of random sequence, the SELEX method includes the steps of contacting the library with the target under favorable conditions for binding, separating unbound nucleic acids from those nucleic acids that have specifically bound molecules target, dissociation of the target nucleic acid complexes, amplification of the nucleic acid dissociated from the target nucleic acid complexes to yield an enriched library of nucleic acid ligand, and then reiterate the steps of binding, separation, dissociation and amplification through as many cycles as desired, to yield highly specific high-affinity nucleic acid ligands for the target molecule.

[00110] SELEX is based on the principle that within a nucleic acid library containing a large number of possible sequences and structures, there is a wide range of binding affinities for a given target. A nucleic acid library comprising, for example, a random 20 nucleotide segment may have structural possibilities of the order of 420. Those that have the highest affinity constants for the target are considered the most likely to bind. The separation, dissociation and amplification process generates a second nucleic acid library, enriched for candidates with greater binding affinity. Additional selection cycles progressively favor the best ligands until the resulting library is predominantly composed of just one or a few strings. These can then be cloned, sequenced and individually tested for binding affinity as pure ligands.

[00111] Selection and amplification cycles are repeated until a desired objective is achieved. In the most general case, the selection / amplification continues until no significant improvement in binding power is achieved in the repetition of the cycle. The iterative selection / amplification method is sensitive enough to allow isolation of a single variant in the sequence in a library containing at least 1011 sequences. The method can, in principle, be used to sample as many as about 1018 different nucleic acid species. Nucleic acids from the library preferably include a portion of the randomized sequence, as well as conserved sequences necessary for efficient amplification. Nucleic acid sequence variants can be produced in a number of ways, including randomized nucleic acid sequence synthesis and size selection of randomly cleaved cellular nucleic acids. The variable sequence part may contain a total or partially random sequence, it may also contain sub-parts of the conserved sequence incorporated with the randomized sequence. Sequence variation in the test nucleic acids can be introduced or increased by mutagenesis before or during the selection / amplification iterations and by specific modification of the cloned aptamers.

Antibodies.

[00112] B-RAf polypeptides or peptides derived therefrom, can be used to generate antibodies for use in the present invention. The B-Raf peptides used preferably comprise an epitope that is specific for a mutant B-Raf polypeptide according to the invention. Polypeptide fragments that function as epitopes can be produced by any conventional means (see, for example, US 4,631,311). In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and more preferably between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid residues in length.

[00113] Antibodies can be generated using antigenic B-Raf polypeptide epitopes according to the invention by immunizing animals such as rabbits or mice, with peptides either free or coupled to the vehicle, for example, by intraperitoneal and / or intradermal injection of emulsions containing about 100 pg of peptide or carrier protein and Freund's adjuvant or any other adjuvants known to stimulate an immune response. Several booster injections may be necessary, for example, at intervals of about two weeks, to provide a useful anti-peptide antibody titer that can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in the serum of an immunized animal can be increased by selecting anti-peptide antibodies, for example, by adsorption to the peptide on a solid support and elution of the antibodies selected according to methods known in the art. .

[00114] The B-RAf polypeptides of the present invention, and their immunogenic and / or antigenic epitope fragments can be fused to other polypeptide sequences. For example, the polypeptides of the present invention can be fused to immunoglobulin domains. Chimeric proteins consisting of the first two domains of the CD4 polypeptide and several domains of the regions contained in mammalian immunoglobulin heavy or light chains have been shown to have advantageous properties in vivo (see, for example, EP 0394827, Traunecker et al., (1988) Nature, 331: 84-86). Increased release of an antigen through the epithelial barrier to the immune system has been demonstrated for antigens (such as insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, for example, WO 96/22024 and WO 9/04813 ).

[00115] In addition, the polypeptides of the present invention can be fused to marker sequences, such as a peptide that facilitates the purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the label provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311) among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci USA 86: 821-824 (1989), for example, hexahistidine provides convenient purification of the fusion protein. Another tag peptide useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., (1984) Cell 37: 767. Thus, any of these fusions above can be moneyed using acids nucleic acids or polypeptides of the present invention.

[00116] In a preferred embodiment, the invention provides antibodies that recognize, specifically B-RAf mutants as described herein.

[00117] Antibodies as described here, are especially indicated for diagnostic application. Therefore, they can be altered antibodies comprising an effector protein as a label. Particularly preferred are labels that allow imaging of the antibody distribution in vivo. Such labels can be radioactive labels or radio-opaque labels, such as metallic particles, which are readily visualized within a patient's body. In addition, they can be fluorescent labels or other labels that are visible on the fabric.

[00118] Recombinant DNA technology can be used to improve the antibodies of the invention. Thus chimeric antibodies can be constructed in order to reduce their immunogenicity in diagnostic or therapeutic applications. In addition, immunogenicity can be minimized by humanizing the antibodies by CDR grafting [see European Patent Application 0 239 400 (Winter)] and, optionally, modifying the framework [EP 0 239 400; Riechmann, L. et al., Nature, 332, 323-327, 1988; Verhoeyen M. et al., Science 239, 1534-1536, 1988; Kettleborough, C. A. et al., Protein Eng. 4, 773-783, 1991, Maeda, H. et al., Human Antibodies and Hybridoma, 2, 124-134, 1991, Gorman S. D. et al., Proc. Natl. Acad. Sci. USA, 88, 4181-4185, 1991; Tempest P. R. et al., Bio / Technology, 9 266-271, 1991; Co, M. S. et al., Proc. Natl. Acad. Sci. USA, 88, 2869-2873, 1991; Carter, P. et al., Proc. Natl. Acad. Sci USA, 89, 4285-4289, 1992; Co, M. S. et al., J. Immunol., 148, 1149-1154, 192 and Sato, K. et al., Cancer Res., 53, 851-856, 1993].

[00119] Antibodies as described herein, can be produced in cell culture. Recombinant DNA technology can be used to produce antibodies according to the established procedure, in bacterial culture, or preferably, mammalian cell culture. The selected cell culture system optionally secretes the antibody product, although antibody products can be isolated from non-secreting cells.

[00120] Accordingly, the present invention includes a process for producing an antibody according to the invention comprising culture of a host, for example, E. coli an insect cell or a mammalian cell, which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoded a signal peptide linked in the reading frame appropriate to a second DNA sequence encoding such antibody protein, and isolating said protein.

[00121] The multiplication of hybridoma cells or mammalian host cells in vitro is performed in a suitable culture medium, which is the customary standard culture medium, for example, Dulbeco's Modified Eagle Medium (DMEM) or RPMI 1640 medium , optionally substituted by a mammalian serum, for example, fetal bovine serum or trace elements and growth support supplements, for example, feeder cells such as normal mouse peritoneal exudate cells, spleen cells, spinal cord macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid or similar. The multiplication of host cells that are bacterial cells or yeast cells is similarly carried out in a suitable culture medium, known in the art, for example, for bacteria in the medium LB NZCYM, NZYM, NZM, Broth Terrific, SOC, SOC, 2 x YT, or M9 Minimum Medium and for yeast in YPD, YEPD, Minimum Medium or Passage Medium.

[00122] In vitro production provides relatively pure antibody preparations and allows for steps giving large amounts of the desired antibodies. Techniques for culturing bacterial, yeast or mammalian cells are known in the art and include homogeneous suspension culture, for example, in an air-suspended reactor or in a continuous agitation reactor, or immobilized or trapped cell culture, for example, in hollow fibers, microcapsules or micro-pearls of agarose or ceramic cartridges.

[00123] Large quantities of the desired antibodies can also be obtained for multiplication of mammalian cells in vivo. To this end, hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause the development of antibody-producing tumors. Optionally, animals are started with a hydrocarbon, especially mineral oils, such as pristane (tetramethyl-pentadecane) before injection. After one to three weeks, the antibodies are isolated from the body fluids of these mammals. For example, hybridoma cells obtained by fusing suitable myeloma cells with antibody-producing spleen cells from Balb / c mice or transfected cells derived from the Sp2 / 0 hybridoma cell line that produce the desired antibodies are injected intraperitoneally into Balb mice / c, optionally pre-treated with pristane, and then, one to two weeks later, the ascitic fluid (abdominal cavity) is removed from the animals.

[00124] The preceding technique and others are discussed, for example, in Kohler and Milstein, (1975) Nature 256: 495-497, US 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual, (1988), Cold Spring Harbor, now incorporated by reference. Techniques for preparing recombinant antibody molecules are described in the references above and also, for example, EP 0623679; EP 0368684 and EP 0436597, which are hereby incorporated by reference.

[00125] Cell culture supernatants are examined for the desired antibodies, preferably by an enzyme immunoassay, for example, a sandwich assay or a dot assay or a radio immunoassay.

[00126] For the isolation of antibodies, the immunoglobulin in the culture supernatant or in the ascitic fluid can be concentrated, for example, by precipitation with ammonium sulfate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes or similar. If necessary and / or desired, antibodies are purified by customary chromatography methods, for example, gel filtration, ion exchange chromatography, DEAE-cellulose chromatography and / or (immuno) -affinity chromatography, for example, affinity with the target antigen, or with Protein-A.

[00127] The invention also relates to hybridoma cells secreting the monoclonal antibodies of the invention. Preferred hybridoma cells of the invention are genetically stable, secrete monoclonal antibodies of the invention of desired specificity and can be activated from deeply frozen cultures by thawing and recloning.

[00128] The invention in a preferred embodiment, relates to the production of anti-mutant B-Raf antibodies. Thus, the invention also relates to a process for preparing a hybridoma cell line secreting monoclonal antibodies according to the invention, characterized by the fact that a suitable mammal, for example, a Balb / c mouse is immunized with one or more PDGF polypeptides or antigenic fragments thereof, or an antigenic vehicle containing a mutated B-Raf polypeptide, immunized mammalian antibody-producing cells, fused with cells from an appropriate myeloma cell line, hybrid cells obtained in the fusion are cloned, and the cell clones secreting the desired antibodies are selected. For example, spleen cells from Balb / c mice immunized with mutant B-RAf are fused with myeloma cell line PAI or the myeloma cell line Sp2 / 0-Ag14 the hybrid cells obtained are examined for secretion of the desired antibodies, and the hybridoma positive cells are cloned.

[00129] A process for preparing a hybridoma cell line is preferred, characterized by the fact that Balb / c mice are immunized by subcutaneous and / or intraperitoneal injection between 1 and 100 pg of mutant B-RAf and a suitable adjuvant , as Freund's adjuvant, several times, for example, four to six times, for several months, for example, between two and four months, and spleen cells from immunized mice are removed two to four days after the last injection and fused with cells of the PAI myeloma cell line, in the presence of a fusion promoter, preferably polyethylene glycol. Preferably, myeloma cells are fused with a three to twenty-fold excess of spleen cells from mice immunized in a solution containing about 30% to about 50% polyethylene glycol of a molecular weight of around 4000. After fusion, the cells are expanded in a suitable culture medium as described above, supplemented with a selection medium, for example, HAT medium at regular intervals, in order to prevent the normal myeloma cells from overdeveloping the desired hybridoma cells.

[00130] The invention also relates to recombinant nucleic acids comprising an insert encoding a heavy chain variable domain and / or a light chain variable antibody targeting mutant B-RAf as described above. By definition such DNAs comprise encoding single-stranded DNAs, double-stranded DNAs, consisting of such encoding DNAS, and complementary DNAs thereto, or these complementary DNAs (single stranded).

[00131] In addition, DNA encoding a heavy chain variable domain and / or an antibody light chain variable domain targeting a mutant B-Raf can be enzymatically or chemically synthesized DNA with the authentic DNA sequence encoding a heavy chain variable domain and / or for a light chain variable domain or a mutant thereof. An authentic DNA mutant and a DNA encoding a heavy chain variable domain and / or a light chain variable domain of the above-mentioned antibodies in which one or more amino acids are deleted or exchanged with one or more other amino acids. Preferably such modifiers are outside the heavy chain variable domain and / or antibody light chain variable domain CDRs. Such a mutant DNA is also intended to be a mutant mutant where one or more nucleotides are replaced with other nucleotides, with the new codons encoding the same amino acids. Such a mutant sequence is also a degenerate sequence. Degenerate sequences are degenerated within the meaning of the genetic code by the fact that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change in the originally encoded amino acid sequence. Such degenerate sequences may be useful, due to their different restriction sites and / or frequency of particular codons that are preferred by the specific host, particularly E. coli, to obtain an optimal expression of the heavy chain murine variable domain and / or a light chain murine variable domain.

[00132] In this context, the term mutant is intended to include mutant DNA obtained by in vitro mutagenesis of authentic DNA according to methods known in the art.

[00133] For the assembly of the complete tetrameric immunoglobulin molecules and expression of chimeric antibodies, the recombinant DNA inserts coding for variable and heavy and light chain domains are fused with the corresponding DNAs coding for constant heavy and light chain domains, the then transferred to appropriate host cells, for example, after incorporation into hybrid vectors.

The invention therefore also relates to recombinant nucleic acids comprising an insert encoding a heavy chain murine variable domain of an anti-mutant B-Raf antibody fused to the human constant domain y, for example, y1, y2, y3 or y4, preferably y1 or y4. Similarly, the invention relates to recombinant DNAs comprising an insert encoding a light chain murine variable domain of an anti-mutant B-Raf antibody targeting the mutant B-Raf fused to a human K or X constant domain, preferably K.

[00135] In another embodiment, the invention is pertinent to recombinant DNAs encoding a recombinant polypeptide where the heavy chain variable domain and the light chain variable domain are linked by means of a spacer group, optionally comprising a signal sequence facilitating the processing of the antibody in the host cell and / or a DNA encoding a peptide that facilitates the purification of the antibodies and / or a cleavage site and / or a peptide spacer and / or an effector molecule.

[00136] Antibodies and antibody fragments according to the invention, are useful in diagnosis. Therefore, the invention provides a composition for diagnosis comprising an antibody according to the invention.

[00137] In the case of a diagnostic composition, the antibody is preferably provided together with means for detecting the antibody, which may be enzymatic, fluorescent, radioisotope or other means. The antibodies and the means of detection can be provided for simultaneous, simultaneous, separate or sequential use, in a diagnostic kit for diagnosis.

[00138] The antibodies of the invention can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blot, radioimmunoassays, ELISA, sandwich immunoassays, immunoprecipitation assay, precipitin reactions, precipitin reactions gel diffusion, immunodiffusion assays, agglutination assays, complement-fixation assays, assays, immuno-radiometrics, fluorescent immunoassays and protein A immunoassays. Such assays are routine in the art (see, for example, Ausubel et. al. eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is hereby incorporated by reference in its entirety). Exemplary immunoassays are briefly described below.

[00139] Immunoprecipitation protocols generally comprise the lysis of a cell population in a lysis buffer as a RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, NaCl 0.15M, 0.01M sodium phosphate at pH 7. Trasilol 2.15) supplemented with protein phosphatase and / or protease inhibitors (eg EDTA, PMSF, aprotinin, sodium vanadate) adding the antibody of interest to the lysate cell, incubating for a period of time (eg 1-4 hours) at 4 ° C, adding beads of protein A and / or protein G sepharose to the cell lysate, incubating for about an hour or more at 4 ° C, wash the beads in lysis buffer and resuspend the beads in SDS / sample buffer. The ability of the antibodies and interest in immunoprecipitating a particular antigen can be assessed, for example, by western staining analysis.

[00140] Western staining analysis generally comprises preparing protein samples, electrophoresis of protein samples on a polyacrylamide gel (eg 8% -20% SDS-PAGE depending on the molecular weight of the antigen) transferring the protein sample from the gel polyacrylamide for a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution, (for example, PBS with 3% BSA or skim milk), washing the membrane in wash buffer (for example, PBS-Tween 20) , expose the membrane to a primary antibody (the antibody of interest) diluted in blocking buffer, wash the membrane in wash buffer, expose the membrane to a secondary antibody (which recognizes the primary antibody, for example, an anti-human antibody ) conjugated to an enzymatic substrate (eg horse radish peroxidase or alkaline phosphatase) or radioactive molecule (eg P32 or I125) diluted in blocking buffer, washing the membrane in washing buffer, and detecting as to the presence of the antigen.

[00141] ELISAs comprise the preparation of the antigen, coating the reservoir of a 96 well microtiter plate with the antigen, adding the antibodies of interest conjugated to a detectable compound such as an enzymatic substrate (eg horse radish peroxidase or phosphatase alkaline) to the reservoir and incubate for a period of time detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound, on the contrary, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound, can be added to the compartment. In addition, instead of covering the reservoir with the antigen, the antibodies can be coated on the reservoir. In this case, a second antibody conjugated to the detectable compound can be added following the addition of the antigen of interest to the coated reservoir.

[00142] The binding affinity of an antibody to an antigen and the extra coefficient of an antibody-antigen interaction can be determined by competitive binding assays. An example of a competitive binding assay is a radioimmunoassay comprising incubating the labeled antigen (e.g., H3 or I125) with the antibodies of interest in the presence of increasing amounts of unlabeled antigen, and detecting the antibodies bound to the labeled antigen. The affinity of the antibodies of interest for a given antigen and the extra binding coefficients can be determined from the data by analysis on an improvised plotter. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with the antibodies of interest conjugated to a labeled compound (for example, H3 or I125) in the presence of increasing amounts of a second unlabeled antibody.

Preparation of mutant B-Raf polypeptides

[00143] Mutant B-Raf polypeptide according to the present invention can be produced by any desired technique, including chemical synthesis, isolation of biological samples and expression of a nucleic acid encoding such a polypeptide. Nucleic acids, in turn, can be synthesized or isolated from biological sources of mutant B-Raf.

[00144] The invention thus relates to vectors encoding a polypeptide according to the invention, or a fragment thereof. The vector can be, for example, a phage, plasmid, viral or retroviral vector.

[00145] Nucleic acids according to the invention can form part of a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or into a complex with a charged lipid. If the vector is a virus, it can be packed in vitro using an appropriate conditioned cell line, and then taken to host cells by transduction.

The nucleic acid insert is operably linked to an appropriate promoter, such as the lambda PL phage promoter, the E. coli lac, trp, phoA and tac promoters, the primitive and late SV40 promoters and retroviral LTR promoters. Other suitable promoters are known to those skilled in the art. The expression constructors also contains sites for initiation of transcription, termination and, in the transcribed region, a ribosome binding site for translation. The coding part of the transcripts expressed by the constructors, preferably includes a translation initiation codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

[00147] As indicated, expression vectors preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or resistance to neomycin for eukaryotic cell culture and tetracycline, genes resistant to kanamycin or ampicillin for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyes and Salmonella typhimurium cells, fungal cells, such as yeast cells (for example, Saccharomyces cerevisiae or Pichia pastoris), insect cells such as Drosophila S2 and Spodoptera Sf9, animal cells like CHO, COS, 293 and Bowes melanoma cell, and plant cells.

[00148] Appropriate culture medium and conditions for the host cells described above are known in the art and are commercially available.

[00149] Among preferred vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN inc., PBluescript vectors, Phagescript, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc. and ptrc99a, pKK2233, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Preferred eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene, and pSVK3, pBPV, pMG and pSG. preferred expression vectors for use in systems and yeast include, but are not limited to, pYES2, pYDl, pTEFl / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3,5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K and PA0815 (all available from Invitrogen, Carlsbad, CA).

[00150] The introduction of the constructor into the host cell can be performed by transfection of calcium phosphate, transfection mediated by DEAE-dextran, transfection mediated by cationic lipid, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Sambrook et al., Referred to above.

[00151] A polypeptide according to the invention can be recovered and purified by recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, interaction chromatography hydrophobic, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is used for purification.

[00152] Polypeptides according to the present invention can also be recovered from biological sources, including bodily fluids, tissues and cells, especially cells derived from an individual's suspected tumor or tumor tissue.

[00153] Furthermore, the polypeptides according to the invention can be chemically synthesized using known techniques (for example, see Creighto, 1983, Proteins: Structures and Molecular Principles, WH Freeman & co., NY ad Hunkapiller et al., Nature , 310: 105-111 (1984)). For example, a polypeptide corresponding to a fragment of a mutant B-Raf polypeptide can be synthesized using a peptide synthesizer.

B-Raf mutations

[00154] Mutations in B-Raf have been identified in human tumor cells. Table 1 describes the location of these mutations and the tumors in which they were identified. The mutations are in the B-Raf kinase domain. Most mutations can be confirmed as somatic, indicating that a similar normal cell / tumor sample has been tested and the mutation found only in the tumor sample. TABLE 1

Compound Assays

[00155] According to the present invention mutant B-Raf is used as a target to identify compounds for example, conductive compounds for pharmacists who are able to modulate the proliferative activity of mutant B-Raf. Therefore, the invention relates to an assay and provides a method for identifying a compound or compounds capable, directly or indirectly, of modulating the activity of mutant B-Raf, comprising the steps of: (e) incubating mutant B-Raf with the compound or compounds for evaluation, and (f) identify the compounds that influence the activity of the mutant b-RAf.

[00156] Mutant B-RAf is as indicated in the context of the present invention.

[00157] According to a first embodiment of this inventive aspect, the assay is configured to detect polypeptides that bind directly to the B-Raf mutant.

[00158] The invention therefore provides a method for identifying a modulating cell proliferation, comprising the steps of (g) incubating mutant B-Raf with the compound or compounds for evaluation and (h) identifying the compounds that bind to Mutant B-Raf. Preferably, the method further comprises the step of: (i) evaluating compounds that bind to the mutant B-Raf for the ability to modulate cell proliferation in a cell-based assay.

[00159] Binding to the B-RAf mutant can be assessed by any technique known to those skilled in the art. Examples of suitable assays include the two hybrid assay systems, which measure in vivo interactions, affinity chromatography assays, for example, involving binding to polypeptides immobilized on a column, fluorescence assays, in which the binding of compounds and mutant B -Raf is associated with changes in the fluorescence of one or both components in a bonding pair and the like. Preferred assays are performed in vivo on cells, such as the hybrid double assay.

[00160] In a preferred aspect of this embodiment, the invention provides a method for identifying a conductive compound for a drug useful in the treatment of diseases involving or using cell proliferation, comprising incubating a compound or compounds to be tested with the B-RAf mutant , under conditions, in which, simply by the presence of the test compound or compounds, B-Raf associates with RAS with a reference affinity, to determine the binding fineness of the B-Raf mutant for RAS in the presence of the compound or compounds for test, and selects compounds that modulate the binding affinity of the B-Raf mutant for RAS with respect to the reference binding affinity.

[00161] Preferably, therefore, the assay according to the invention is calibrated in the absence of the test compound or compounds, or in the presence of a reference compound whose activity in binding to the B-RAf mutant is known, or is from another desirable as a reference value. For example, in a double-hybrid system, a reference value can be obtained in the absence of any compound. The addition of a compound or compounds that increase the binding affinity of the B-Raf mutant to a target increases the assay conference above the reference level, while the addition of a compound or compounds that reduce this affinity results in a reduction of test conference below the reference level.

[00162] In a second embodiment, the invention can be configured to detect functional interactions between a compound or compounds and the B-Raf mutant. Such interactions will occur, either at the level of mutant B-Raf regulation, such that this kinase is itself activated or inactivated, for example, by RAS, in response to the test compound or compounds, or at the level of the modulation of the biological effect of the Mutant B-Raf on downstream targets such as MEK. As used herein, "activation" and "inactivation" includes the modulation of activity, enzymatic, or otherwise, of a compound, as well as the modulation of its production rate, for example, by activating or suppressing the expression of a polypeptide in a cell. The terms include direct action on the genetic transcription of were to modulate the expression of the gene product.

[00163] Tests that detect the modulation of the functional interaction between B-Raf downstream and its partners upstream or downstream in a signaling step are preferably cell-based tests. For example, they can be based on an assessment of the degree of MAPK phosphorylation, which is indicative of the degree of MEK activation, resulting from the activation of the mutant B-RAf.

[00164] In preferred embodiments, a nucleic acid encoding mutant B-Raf is linked to a vector, and introduced into suitable host cells to produce transformed cell lines that express mutant B-RAf. The resulting cell lines can then be produced by reproducible qualitative and / or quantitative analysis of the effects of potential compounds affecting the function of mutant B-Raf. Thus, cells expressing mutant B-Raf can be used to identify compounds, particularly low molecular weight compounds, that modulate the function of the mutant B-Raf. Thus, host cells expressing mutant B-raf are useful for drug selection and it is a gold object of the present invention to provide a method for identifying compounds that modulate the activity of the mutant B-Raf, such a method comprising exposing cells containing heterologous DNA encoding B -Rafant mutant, where such cells produce functional mutant B-RAf, to at least one compound or mixture of compounds or signal whose ability to modulate the activity of such mutant B-RAf seeks to determine and then monitor such cells for changes caused by such modulation. Such an assay allows the identification of modulators, such as agonists, antagonists and allosteric modulators, or mutant B-RAf. As used herein, a compound or signal that modulates the activity of the mutant B-RAf refers to a compound that alters the activity of the mutant B-Raf in such a way that the activity of the mutant B-RAf in a target thereof, like MEK, it is different in the presence of the compound or signal (if compared to the absence of such a compound or signal).

[00165] Cell-based selection assays can be designed by constructing cell lines in which the expression of a reporter protein, that is, an easily testable protein, such as p-galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase, it is dependent on the activation of a mutant B-RAf substrate. For example, a reporter gene encoding one of the above polypeptides can be placed under the control of a response element that is specifically activated by MEK or MAPK. Such an assay allows the detection of compounds that directly modulate the mutant B-RAf function, such as compounds that antagonize MEK phosphorylation by mutant B-Raf, or compounds that inhibit or potentiate other cellular functions required for B-Raf mutant activity. The cells in which non-mutant wild-type B-Raf is present provide adequate controls.

[00166] Alternatively, assay formats include assays that directly evaluate proliferative responses in a biological system. Constitutive expression of unregulated mutant B-RAf results in a proliferative phenotype in animal cells. Cell-based systems, such as 3T3 fibroblasts, can be used to assess the activity of potential regulators of mutant B-Raf.

[00167] In a preferred aspect of this embodiment of the invention, a method is provided for identifying a reference compound for a pharmacist useful in the treatment of disease involving or using an inflammatory response, comprising; incubation of a test compound or compounds with mutant B-RAf and MEK, under conditions where, solely by the presence of the test compound or compounds, mutant B-Raf directly or indirectly causes MEK phosphorylation with a reference phosphorylation efficiency ; determine the ability of the mutant B-Raf to cause the phosphorylation, directly or indirectly, of MEK in the presence of the compound or compounds to be tested, and selection of compounds that modulate the ability of the mutant B-RAF to phosphorylate MEK with respect to the phosphorylation efficiency of reference.

[00168] In a further preferred aspect, the invention relates to a method for identifying a reference compound for a pharmacist, comprising the steps of: providing a purified mutant B-Raf molecule; incubating the mutant B-Raf molecule with a substrate known as phosphorylated by mutant B-Raf and a test compound or compound, and identifying the test compound or compounds capable of modulating substrate phosphorylation.

[00169] A substrate for phosphorylation of mutant B-Raf is MERK. Preferably, therefore, MEK is used as a substrate to monitor compounds capable of modulating the mutant B-Raf kinase activity. This facilitates those skilled in the art to select directly for kinase modulators. Preferably kinase modulators are kinase inhibitors (mutant B-Raf).

[00170] In a preferred embodiment, the activity of BRaf can be measured according to the following protocol:

[00171] 1. The cells are solubilized in lysis buffer (150 mM NaCl, 25 mM HEPES [Ph 7.3], 1 Mm sodium orthovanadate, 1% Triton X-100, protease inhibitors, dithiothreitol).

[00172] 2. The lysate is incubated on ice for 10 minutes and centrifuged at 14,000 3 g for 10 minutes, and the supernatant incubated with polyclonal anti-B-Raf antibodies, and then with protein G-Sepharose at 4 ° C for 1 hour.

[00173] 3. The immunoprecipitates are washed twice with lysis buffer, and the kinase reaction performed at 30 ° C for 10 minutes in kinase buffer (0.2 mM ATP, 30 mM MgCl2, 2 mM MnCl2, 40 mM p -sodium glycerophosphate, 0.2 mM ortho-vana-sodium date, 2 mM ocadic acid, 0.2% p-mercaptoethanol) with 1 mg of purified recombinant MEK1 added as the substrate.

[00174] 4. After activation of MEK1, 15 mCi of ATP [rp32] and 1 mg of defective kinase (K52R) ErK are added as the substrate for another 2 minutes. The reaction ends with the addition of sample buffer, the mixture being boiled for 5 minutes and the proteins separated by SDS-PAGE.

[00175] 5. The gel proteins are transferred to a polyvinylidene difluoride membrane in which the amount of radio-labeled K52R ERk is quantified by a PhosphorImager.

[00176] 6. For calculations of B-Raf activity, the amount of B-Raf protein on the same membrane is determined by a membrane probe with I125-labeled goat anti-mouse IgG following monoclonal anti-B-Raf staining of mouse.

[00177] 7. The assay can be repeated in the presence or absence of the test compound (s).

[00178] Optionally, the identified test compound (s) can then be subjected to in vivo testing to determine its effectiveness on a mutant B-Raf signaling pathway, for example, as indicated in the preceding modality

[00179] As used herein, "mutant B-Raf activity" can refer to any activity of the mutant BRaf, including its binding activity, in particular it refers to the phosphorylation activity of the mutant B-Raf. Therefore, the invention can be configured to detect the phosphorylation of the target compound (s) by mutant B-Raf, and the modulation of this activity by potential therapeutic agents.

[00180] Examples of compounds that modulate the phosphorylation activity of the mutant B-RAf, include dominant negative mutants of the B-Raf itself. Such compounds are able to compete for the mutant B-Raf target, thereby reducing the activity of the mutant B-Raf in a biological or artificial system. Thus, the invention furthermore relates to compounds capable of modulating the phosphorylation activity of the mutant B-Raf.

[00181] Compounds that influence mutant B-Raf activity can be of almost any generic description, including low molecular weight compounds, including organic compounds that can be linear, cyclic, polycyclic or a combination thereof, polypeptide peptides including antibodies , or proteins. In general, as used herein, "peptides", "polypeptides" and "proteins" are considered equivalent.

[00182] Many compounds according to the present invention can be conductive compounds useful for drug development. Useful conducting compounds are especially antibodies and peptides, and particularly, intracellular antibodies expressed within the cell in the context of gene therapy, which can be used as a model for the development of peptide or low molecular weight therapies. In a preferred aspect of the invention, conductive compounds and mutant B-Raf or other target peptides can be co-crystallized in order to facilitate the design of suitable low molecular weight compounds that mimic the observed interaction with the conductive compound.

[00183] Crystallization involves the preparation of a crystallization buffer, for example, mixing a solution of the peptide or peptide complex with a "reservoir buffer" preferably in a 1: 1 ratio with a lower concentration of the precipitating agent necessary for crystal formation. The formation of the crystal, the concentration of the precipitating agent is increased, for example, by adding the precipitating agent, for example, by titration or by allowing the concentration of the precipitating agent to be equilibrated by diffusion between the crystallization buffer and a buffer reservoir. Under suitable conditions such diffusion of the precipitation agent occurs along the gradient of the precipitation agent, for example, from the reservoir buffer with a higher concentration of precipitation agent to the crystallization buffer with a lower concentration of the precipitation agent. Diffusion can be achieved, for example, by steam diffusion techniques that allow diffusion in the common gas phase. Known techniques are, for example, steam diffusion methods, such as the "drop drop" or "seated drop" method. In the steam diffusion method, a drop of the crystallization buffer containing the protein hangs above or sits next to a much larger reservoir buffer tank. Alternatively, equilibration of the precipitating agent can be achieved through a semipermeable membrane that separates the crystallization buffer from the reservoir buffer and prevents dilution of the protein into the reservoir buffer.

[00184] In the crystallization buffer the peptide or peptide / binding partner complex preferably has a concentration of up to 30 mg / ml, preferably from about 2 mg / ml to about 4 mg / ml.

[00185] The formation of crystals can be acquired under varied conditions that are essentially determined by the following parameters: pH, presence of salts and additives, precipitating agent, concentration and temperature of the protein. The pH can vary from about 4.0 to 9.0. The concentration and type of buffer is not important, and therefore variable, for example depending on the desired pH. Suitable buffer systems include phosphate, acetate, citrate, Tris, MES and HEPES. Useful salts and additives include, for example, chlorides, sulfates, and other salts known to those skilled in the art. The buffer contains a precipitating agent selected from the group consisting of a water-miscible organic solvent, preferably polyethylene glycol with a molecular weight between 100 and 20000, preferably between 4000 and 10,000, or a suitable salt, such as sulphates, particularly ammonium sulfate, a chloride, a citrate or a tartrate.

[00186] A crystal of a peptide or peptide / binding partner complex according to the invention can be chemically modified, for example, by heavy atom derivatization. In summary, such derivatization can be acquired by soaking a crystal in a solution containing heavy metal atom salts, or an organometallic compound, for example, lead chloride, gold thiomalate, thimerosal or uranyl acetate, which is capable of diffuse through the crystal and attach to the surface of the protein. The locations of the attached heavy metal atoms can be determined by X-ray diffraction analysis of the soaked crystal, the information of which can be used, for example, to build a three-dimensional model of the peptide.

[00187] A three-dimensional model can be obtained, for example, from a heavy atom derivative of a crystal and / or from all or part of the structural data provided by the crystallization. Preferably, the construction of such a model involves homology modeling and / or molecular substitution.

[00188] The preliminary homology model can be created by combining sequence alignment with any RAF kinase whose structure is known, secondary structure prediction and selection of structural libraries. For example, the sequences of the mutant B-Raf and a candidate peptide can be aligned using a suitable software program.

[00189] Computational software can also be used to predict the secondary structure of the peptide or peptide complex. The peptide sequence can be incorporated into the mutant B-Raf structure. Structural inconsistencies, for example, structural fragments around insertions / deletions, can be modeled by selecting a structure library for peptide of desired length and with an adequate conformation. To predict the side chain conformation, a side chain rotamer library can be employed.

[00190] The final homology model is used to solve the crystal structure of the peptide by molecular substitution using appropriate computer software. The homology model is positioned according to the results of molecular substitution, and subjected to further refinement comprising dynamic molecular calculations, and modeling of the inhibitor used for crystallization in electron density.

Kinase activation studies

[00191] Constitutively active kinase mutants are valuable research tools for understanding signaling pathways and developing therapeutic agents that modulate such pathways. The activity of five of the mutants according to the invention was examined. These are G463V, G468A, G955R, L596V and V599E. To examine the activity of the mutants, versions labeled smyc-epitope of B-Raf are temporarily expressed in COS cells. In order to examine the activity of this exogenously expressed B-RAf, the protein is immunoprecipitated using the myc-tag and examined in a cascade kinase assay, using GST-MEK, GST-ERK bacterially produced and myelin basic protein (MBP) as sequential substrates (Marais et al (1997); J. Biol. Chem 272: 437883). B-RAf has high levels of basal kinase activity and is significantly more active in the absence of activators than either Raf-1 or A-Raf (Marais et al (1997); J. Biol. Chem 272: 4378-83). In addition, while Raf-1 and A-Raf require both Ras oncogenic (RasV12) and activated Src to stimulate their activity completely, B-Raf is completely activated by co-expression with RasV12 alone. The effect that these mutations have on both baseline B-Raf activity and activity stimulated by Rasv12 is therefore tested. Compared to B-Raf wild type B- RafG463V, B-RafG468A, B-RafL596V and B-RafV599E all have strongly elevated basal kinase activity (Figure 1A, 1B). By comparison, G-RafG595R reduced baseline activity compared to wild-type protein (Figure 1a). Similar results are seen in vivo. All five mutants are stimulated by oncogenic Ras (RasV12). However, double activation for each of the mutants is reduced compared to wild-type B-Raf (Figure 1A, B) and is particularly small in the case of B-RAf V599E. However, since the basal activity of each of B-RAfG463V, B-RafG468A, B-RafL596V and B-RafV599E is greater than the wild type protein, the absolute levels of activity seen are higher in each case the presence of RasV12 than for wild type protein. Interestingly, B- RafG595R is also stimulated by RAsV12, but activation was very weak, probably due to low starting levels.

[00192] The ability of each of the activated mutants to transform NIH3T3 cells is also examined. In this assay, wild-type B-Raf transforming cells have very low efficiency (~ 0.02% of the number of colonies seen with RasV12). However, as seen in Figure 2, each of the activated mutants transform NIH3T3 cells 40 to 85 times more efficiently than wild-type B-raf does.

[00193] The extent of developmental dependence of cells containing the B-Raf mutants in the Ras / MEK pathway is investigated. For these studies, two tests are used. The first lends itself to checking if its growth is suppressed by microinjection of the monoclonal antibody Y13-259, an antibody that neutralizes the activity of the cellular ras. The results are shown in Table 2 The data are divided into three groups. The first group has wild-type B-raf and its growth is inhibited (40-100%) by Y13-259. The second group has B-Raf activation mutations and their growth was not inhibited (<15%) by Y13-259. The third group (only one case) contains a cell that has both an activating Ras and an activating B-RAf mutation. Interestingly, the growth of this cell line was inhibited by Y13-259, but this may be due to the growth being dependent on both Ras and B-Raf.

[00194] The second approach is to examine the effects of compound U0126, an MEK1 / 2 inhibitor, the only known substrates for B-Raf. These results demonstrate that the treatment of cells that have activation mutations in B-RAf results in suppression of cell proliferation, when MEK activity is inhibited indicating that the activation of cell signaling by activated B-raf mutants is a target therapeutic. See Table 3. Taken together, the data above shows that:

[00195] 1. There are two classes of B-RAf mutation in human tumors, activation and inactivation mutations.

[00196] 2. Activated versions of B-Raf are able to transform NIH3T3 cells and therefore can be indicated as oncogenes.

[00197] 3. Human tumor cell lines that express activated B-Raf protein are not sensitive to Y13-259, a Ras neutralizing antibody, indicating that their growth is not dependent on RA proteins and is therefore unlikely to respond to compounds targeting Ras proteins. This indicates that activation mutants may overcome the requirement for Ras signals in tumor cells.

[00198] 4. However, its activity is suppressed by compound U0126, indicating that its growth is dependent on the activity of this path, and therefore likely to respond to therapeutic agents aimed at B-Raf activity.

[00199] 5. Since some of the mutations are in the phosphate bonding circuit of the kinase domain (G463, G465, G468) and these amino acids are conserved in all kinases these mutations represent a global and convenient mechanism for activating kinases. This has important implications for the selection of therapeutic people.

The invention, therefore, provides a constitutively active kinase comprising a mutation in the phosphate binding circuit thereof, selected from the group consisting of mutations in one or more positions corresponding to positions 463, 465 and 468 of B-Raf.

[00201] Preferably, the mutation is in one or more positions 463 and 468.

[00202] More preferably they are G463V and G468A.

[00203] Many kinases are identified because they are associated with a specific disease, but their activation mechanisms may not always be fully understood. Constitutively activated mutants as described herein provide a reagent that can be used to select inhibitors without first having to clarify their activation mechanism.

Exemplary kinases include other kinases in the MAP kinase path, such as MEK and ERK and other MAP kinase paths, such as p38, JNK and their upstream kinases. Although something is known about their activation mechanisms, for some it is not known how to activate them by direct mutation. The present invention provides mutants activated for such kinase selection purposes. In addition, kinases that are downstream of MAP kinases, such as p90Rsk, mnk, etc. can also be activated. Although alternative activation mechanisms are known, mutation may be a preferred route in selection assays.

[00205] The invention also covers some known kinases that have no known mechanism of action, such as Lkb1, which is involved in Putz-jegers syndrome and PDK1 kinase which may be constitutively active, but which may still be activable for drug selection. This kinase is involved in insulin signaling, so it can be a useful target for diabetes. Also involved in type II diabetes is the AMP-activated kinase, which again is activated by phosphorylation and is therefore susceptible to activation by mutation.

[00206] Consequently, the invention provides a method for selecting one or more compounds for a kinase inhibiting effect, comprising: (a) preparing a mutant kinase comprising an amino acid substitution, deletion or insertion at one or more of the 463 positions , 465 or 468 as detailed above, (b) exposing the mutant kinase to one or more compounds in the presence of a kinase substrate, and (d) determining the kinase's ability to phosphorylate the substrate in the presence of one or more compounds.

[00207] The kinase phosphorylation activity in the presence of the test compounds is advantageously compared with its activity in the absence of the compounds, a reduction in the basal activity of the mutant kinase is indicative of kinase inhibition by the compounds. For multiple assays a reference level of phosphorylation can be determined for a given assay, and used as a basis for comparison.

[00208] Preferably the kinase is a Raf protein kinase, advantageously it is B-Raf.

[00209] Conversely, constitutively repressed mutants such as B-Raf G595R are useful in the selection of kinase activators.

Confirmation of bRAF as a drug target

[00210] In order to confirm BRAF as a target in cancer, it was first tested whether the growth of cells expressing activated mutant forms of BRAF required the RAF-MEK-ERK step and signaling for growth. To this end, melanoma and colorectal cell lines that harbor mutations in the RAF gene are treated with pharmacological agents that block signaling through this step. Two compounds are tested. One is compound U0126, which is a MEK inhibitor and therefore decouples RAF-ERK signaling in cells (Sebolt-Leopold et al., 1999) BAY 43-9006 has also been tested. This is an inhibitor and RAF proteins (Lyon set al, 2001). The ability of these compounds to block ERK activity was tested in a melanoma cell line WM266,4, which has an aspartic acid substituted by valine at position 599 of the BRAF gene. This is an activating mutation (Figure 3A). These cells also have elevated basal kinase activity as judged using an antibody (ppERK) that only binds to the double phosphorylated activated ERK version. when ppERK antibodies are used for Western staining of WM266.4 cells, a strong signal is seen in the 42-44 kDA region, indicating that ERK has high basal kinase activity in these cells (Figure 3B) however, when cells are treated with U0126 or Bay 43-006, ERK activity is strongly suppressed (Figure 3B). Similar results were obtained using cell A375, a melanoma cell line that houses a V599E mutation in the BRAF gene (Davies et al., 2002). These data demonstrate that RAF and MEK signaling is necessary to maintain high ERK activity in these cells.

[00211] Next, what effect BAY 43-9002 had on the growth of WM266.4 cells was tested and this compound was found to block the growth of these cells with an IC50 of ~ 6.1 pM (Table 4).

[00212] BAY 43-9006 also blocked the growth of colo 829 cells and BE cells in the micromolar range (Table 4). Colo 829 cells are a melanoma cell line that harbors a V599E mutation in the BRAF gene and BE cells are a colorectal line that harbors a G463 mutation in the BRAF gene (Davies et al., 2002) As shown, both of these mutations are active (Davies et al., 2002). Finally, the effects of these inhibitors on DNA synthesis were tested. Incubation of WM-266.4 cells with 10 pM U0126 or 10 pMBAY 43-9006 strongly suppressed DNA synthesis in these cells (Figure 4). These data demonstrate that ERK activation and proliferation in cells that harbor activation mutations in the BRAF gene are dependent on RAF and MEK activities.

[00213] There are three RAF genes in mammalian cells, CRAF (also called RAF-1), ARAF and BRAF. U0126 is a MEK inhibitor and therefore will not be able to distinguish CRAF from BRAF or ARAF signaling. Similarly, BAY 43-9006 can inhibit both CRAF and BRAF, so it will not distinguish between different RAF isoforms. Therefore, in order to determine which RAF isoform signaled to ERK in WM266.4, the cells were treated with small interference RNA probes (siRNA) that are selective for individual RAF isoforms. WM266.4 cells were treated with siRNA probes designed for BRAF specificity, or CRAF or a competitive control that would not recognize any isoforms. The recognition efficiency for CRAF was determined by Western staining. Treatment of WM 266.4 cells with a CRAF-specific siRNA probe resulted in strong suppression of CRAF expression (Figure 5A). Similar results were seen in Colo 82 cells and BE cells (Figure 5a). When WM-266.4 cells were treated with a BRAF-specific siRNA probe, BRAF activity in the cells was strongly suppressed, but no suppression was observed when the cells were treated with competitive control (figure 5B). Similar results were observed in Colo 829 and BE cells (figure 5B).

[00214] The above data shows that siRNA can be used to selectively suppress the expression of the BRAF and CRAF proteins. It was examined, therefore, how the ablation of each of these proteins affected the ERK activity in these cells. When siRNA was used to remove BRAF protein expression in WM-266.4, eRK activity was suppressed in a time-dependent manner (Figure 6). In contrast, ablation of CRAF expression or treatment with competitive siRNA probes did not affect ERK activity (Figure 6). Similar results were obtained in Colo 829 cells (Figure 6). These results demonstrate that, BRAF is the main isoform that signals ERK in melanoma cells that express activated BRAF proteins. CRAF does not appear to signal baseline ERK activity in these cells.

[00215] Finally, we examined how BRAF ablation affected cell growth, examining the effects on apoptosis in WM-266.4 cells. For these studies, the cells were fixed in 70% ethanol, stained with propridium iodide and their cell cycle profiles were examined by fluorescence activated cell classification (FACS). Using this analysis, apoptotic cells appear at the sub-G1 peak. In these cells, spontaneous apoptosis is very low, with less than 1%

[00216] Confirmation of cell reagent appearing at the sub-G1 peak (Figure 7, table 5). When cells are treated with U0126, the proportion of cells at the sub-G1 peak is significantly increased (~ 3.5%, figure 7, Table 5). Similarly, the ablation of BRAF expression using siRNA also increases the number of cells in the sub-G1 peak, while the ablation of CRAF or treatment with competitive control does not increase. The cleavage of PARP, an apoptosis-inducing marker, was also examined. The treatment of cells with BRAF siRNA induced cleavage of PARP, whereas the competitive control did not. These data demonstrate that, when the mutant BRAF protein is removed in melanoma cells, apoptosis is induced.

[00217] In summary, these results demonstrate that in melanoma cell lines that express BRAF-activated mutants, signaling through rAF and MEK is necessary for ERK activation and cell growth. BRAF, instead of CRAF appears to be the main RAF isoform that stimulates eRK activity, and appears to protect cells from apoptosis. These data suggest that BRAF is an important therapeutic target in cells that rely on BRAF signaling for growth and protection from apoptosis.

High Productivity Selection Development and Testing

[00218] An HTS assay has been revealed for the B-raf mutant V599E. In order to evaluate the results, lysate expressing RAf and GST-MKKL reagents were checked for activity by performing a standard coupled assay using GST-ERK2 (competent kinase) and measuring the incorporation of P33-y-phosphate into myelin basic protein (MBP). As seen in figure 8, in the presence of the B-RAf lysate an increase of 16 times in the signal was observed compared to the control lysate (without expression).

Test Platform Confirmation Option 1: Glutathione FlashPlate Coupled Kinase Assay

[00219] Platform Principle: Substrate labeled GST (ERK -2) is captured on inlaid walls with flickering of one (“flashplate”) via glutathione coating. the incorporation of P33-Y-phosphate into the substrate resulted in a measurable scintillation signal.

[00220] The possibility of measuring the incorporation of P33-Y-phosphate in ERK2 (GST-kdERK2) killed by GST-kinase) as a production of B-Raf activity was evaluated in a glutathione flashplate assay. Figure 9 demonstrates that, using the conditions transferred from the reagent confirmation exercise, we are unable to detect the B-Raf dependent incorporation of P33 into GST-kdERK2 using this platform. A p81 filter plate test also proved unsuccessful. As a consequence of the amplification characteristics of this assay and cascade, maintaining a test signal in the absence of the thin test step (ie, phosphorylation of MBP ERK2) would most likely require increased levels of each of the remaining constituents. It was therefore considered appropriate to evaluate the platform based on antibodies before relying on the costly exercise of reagent titrations of B-Raf, MKKI and ERK2 on this radiometric platform.

Option 2: Coupled Kinase Assay and DELFIA format

[00221] DELFIA Assay (Dissociation-Enhanced Lanta-nid Fluorescent Immunoassay) involves measuring ERK2 phosphorylation via binding of a phospho-specific antibody. The B-Raf / GST-MKKI / GST-kdERK2 coupled kinase assay generates phosphorylated GST-kdERK2. A coated anti-GST plate is used to capture GST-kdERK2. A primary antibody is added, which specifically detects phosphorylated ERK2 in Threonine and Tyrosine. A secondary antibody labeled with europium (Eu) is then added. In the presence of intensification solution. the Eu-label dissociates from the antibodies by absorbing at 335 nm and allowing fluorometric detection at an emission wavelength of 620 nm.

[00222] The B-Raf assay was evaluated on this platform using a combination of the reagent confirmation exercise kinase assay and DELFIA assay conditions. Figure 10 shows that in the presence of the B-Raf lysate an increase of 12.2 times in the signal was observed, compared to the control lysate (without expression). The observed signal was entirely dependent on the presence of all three enzyme / substrate components.

[00223] Based on preliminary experiments, the DELFIA platform was selected for development.

Development of the Kinase Assay B-raf lysate

[00224] Three batches of B-RAf lysate were used throughout the procedure. Lot A was used to establish the DELFIA test platform. Lot B was used during the development of the test. For batches B and C, an approximate linear relationship between the amount of lysate and the level of the signal obtained was evident between 0.025 to 0.1 pl / reservoir. The final quantities selected for each batch were based on obtaining sufficient signal opening in the linear range. As a result of these assessments, Lot A was used at 1 p / reservoir (96 reservoirs) and Lots B and C (Exam) were used at 0.1 pl / reservoir (96 reservoirs) and 0.05 pl / reservoirs [384 reservoirs].

Optimization of Antibody Levels

[00225] Initial considerations regarding possible competition from ERK2 and MKKI (both GST-labeled) for glutathione binding sites resulted in primary developmental tests performed using a "pre-binding protocol". These conditions were as follows: • Pre-connection of 100 ng / GST-kdERK2 reservoir to glutathione coated coils from 96 reservoirs. • Addition of B-RAf lysate (Lot A), MKKI (6.5 pg / ml) and 500 pM ATP) in a final volume of 50 pM DKB (see Appendix I) and incubation at 30 ° C for 1 hour.

[00226] This protocol was used to optimize and save the antibody load of the detection system. Titrations of both primary and secondary antibodies were performed to assess the possibility of reducing antibody levels while maintaining a signal for a background ratio of> 10.1. Figures 4 and 5 indicate that 1: 3000 and 1: 1000 were the lowest acceptable concentrations for fosfoERK2 and antibodies labeled with Eu respectively. all subsequent assays were therefore performed using these concentrations of antibodies.

MKKL and ERK2 Level Optimization

[00227] The ability to perform the enzymatic component of this assay "in solution" without a pre-binding step, was investigated to allow the titration of defined concentrations of both MKKL and ERK2. In addition, reducing this assay to a single (homogeneous) mixing of the reagents involved in the kinase would make it more accessible to HTS. Figure 13 illustrates that the homogeneous assay and the pre-connection protocol gave equivalent data. All subsequent tests were performed using the “homogeneous” protocol.

[00228] The titration of ERK-2, while maintaining MKKL levels indicated that the maximum signal was obtained at a ratio of ~ 12: 1 (ERK2: MKKL). In an attempt to save reagents a matrix titration of these components was performed. Using 0.1 pl of B-Raf (batch B), the combination of 6.5 pg / ml GST-MKKL and 80 pg / ml GST-kdERK2 gave the maximum signal and although some reduction in the Raf / MKKL load was possible , it was considered appropriate to maintain the original relationship. This decision was made with the knowledge that other changes in the test parameter (for example, transfer to 384, reduction in ATP load, transfer to automation) can further reduce the signal window. All subsequent tests were therefore performed using 6.5 pg / ml GST-MKKL and 80 pg / ml GST-kdERK2.

Temperature Effect

[00229] The ability to perform this test at room temperature would significantly simplify the eventual HTS process. The test signal at room temperature and 30 ° C was therefore investigated. Based on the results obtained, signal-to-background ratio was acceptable at room temperature and all subsequent tests were performed at room temperature.

Evaluation of the Assay in Plate Format of 384 Reservoirs

[00230] In an attempt to intensify productivity and minimize the use of reagents during selection, the performance of the assay in 384 reservoir format was evaluated. In this format, the assay was performed well, and both standard assays (50 µl end) and reduced reagent volume (25 µl) returned a highly acceptable signal for background ratios. All subsequent development experiments were carried out in a 384 format using reaction volumes of 25 pl.

Concentration Optimization TP

[00231] The ATP concentration of a kinase selection assay has the potential to influence the number and nature of the identified inhibitory compounds. The definition of ATP levels for such an assay is a balance of the following considerations: • use of ATP levels that allow a measurable, consistent test signal window • use of ATP levels low enough to allow the identification of competitive ATP inhibitors • employment of sufficiently high ATP levels, such that competitive ATP agents likely to be ineffective in the context of cellular ATP, are less likely to be detected.

[00232] Kinase selection assays are usually performed at an ATP concentration relative to Km. Deriving Km values for B-Raf and MKKL requires the development of individual assays for each enzyme, instead of the coupled assay developed there. development of such assays undoubtedly facilitated a future understanding of the mode of action of any inhibitory compounds identified in the selection assay.

[00233] For the purpose of defining the ATP load of the selection test, the concentration dependence of the coupled test was determined. The signal generated is maximum and half Maximo at ~ 200 pM and ~ 18 pM ATP, respectively. Further studies indicated that 50 pM was the lowest ATP concentration at which a robust day-to-day test signal was observed (eg, signal: 8000, 2% CV). ATP levels below this (10 pM) resulted in a greater relative variation in the test signal (ie signal: 2500, 10% CV). The final ATP concentration of the selection assay was set to 50 pM to give a signal window large enough to withstand any friction during the transfer of the assay to automation.

Selection Essay Time Course

[00234] It is critical that a selection test is carried out within its linearity period. To determine the extent of the linear phase of the coupled assay, a time lapse was performed in up to 75 minutes. The reaction was linear between 4 and 45 minutes. The observed “delay” period is characteristic of this assay and reflects the time required to accumulate detectable levels of the reaction product. Based on this study, an incubation period of 45 minutes at room temperature was indicated. Final conditions for Assay B-RafV599E DELFIA The summary of the final selection conditions for the assay were as follows: Enzymatic reaction: • 384 wells plate coated with glutathione • B-Raf lysate 0.05 pl • GST-MKKL 6.5 pg / ml • GST-kdERK2 80 pg / ml • ATP 50 pM • incubation at room temperature for 45 minutes • Final volume of 25 pl Detection conditions: • anti-fosfoERK2 antibodies 1: 3000 • anti-mouse antibodies labeled with Eu 1: 100

Development of Automation and Quality Control Confirmation of Selection Lot Reagents

[00235] Using the conditions described above, the tests were performed to compare the batches of selection and assay development of B-Raf, MKKI and ERK-2. In all cases, the reagent selection batch was performed in an equivalent manner when compared to those used for the development of the assay.

Automated B-RafV599E DELFIA Assay Mini Tests

[00236] In preparation for selection, an automated liquid handling strategy was planned for the test. In order to test this system, 'mini-tests' of the automated B-Raf assay were performed using false selection plates (ie, no compound, but containing standard control columns). The data derived from these experiments represent both a measurement of the vigor of the biological test, as well as the precision and consistency of the automation processes involved.

[00237] In summary, each plate contained B-Raf kinase reactions in all reservoirs of Columns 1-22 and columns 23-24 contained in the control reactions. In order to define the quality of the data generated by the automated assay, 4 mini-plate tests using this format were performed in two different days. Within each ‘mini-test a plate was used to set IC50 values for some of the predicted inhibitors in this assay. The inhibitors covered a range of modes of action: Staurrosporine (ATP-competitive kinase inhibitor), SB203580 (competitive Raf ATP inhibitor) and U0126 (non-competitive MKKL inhibitor for ATP).

[00238] The test shows good consistency, but within the plates and between days. The data obtained demonstrate that, the automated assay meets the criteria defined for an in vitro HTS assay of format 384: • Background signal of at least 10: 1 • Z'de> 0.4 • CV row and column <15 %

Esta tabela demonstra a inibição do crescimento de várias linhas de célula com mutações B-Raf. A proporção de inibição (como uma percentagem do numero de células que não incorporam BrdU) está mostrada na última coluna TABELA 3

Esta tabela demonstra a inibição do crescimento celular em células tratadas com o inibidor MEK U0126 TABELA 4 Inibição do crescimento celular por BAY 43-9006.

Linhas celulares foram incubadas na presença de níveis aumentados de BAY 43-9006 e os níveis do crescimento celular foram determinadas por manchamento com sulforodamina B. Os valores IC50 foram determinados por análise de regressão não-linear e estão indicados. TABELA 5 Análise do ciclo celular

Células WM-266,4 foram tratadas com U0126, DMSO, siRNA para BRAF, siRNA para CRAF ou o controle siRNA competitivo. As células foram incubadas por 96 horas e as células foram fixadas e manchadas com iodeto de proprídio para análise do ciclo celular por FACS. A proporção de células em cada fase do ciclo celular está demonstrada.[00239] The inhibitors employed still make the assay valid, generating inhibition dependent on the concentration of Raf / MKKl activity. Of particular importance, is the fact that the standard inhibitors returned the IC50 values within a double range on separate days. These data also indicate that these compounds would be identified as successful when tested at ~ 30 pm (10 pg / ml) in this selection assay. Final Protocol for B-Raf V599E DELFIA automated Enzyme cocktail (final volume 12 l: 0.05 pl of RAf lysate 0.0325 p of GST-MKKI 0.065 pl of GST-kdERK2 1. 3 pl of the pre-placement test compound in a plate on 384 glutathione-coated plates 2. 2. pl of enzyme cocktail added by PlateMatePlu 3. 10 pl of ATP added to Asys 4. Plaque stirred at room temperature for 45 minutes 5. Plate washed with 3 x 80 pl of DELFIA wash buffer (DWB) using ELX405 plate washer 6. 25 pl of anti-phosfoERK2 added by Multidrop 7. Plate shaken at room temperature for 1 hour 8. Plate washed with DWB 3 x 80 pl. 25 pl of anti-mouse antibodies labeled with Eu added by Multidrop 10. Plate shaken for 30 minutes at room temperature 11. Plate washed with DWB at 3 x 80 pl 12. Increment solution DELFIA at 25 pl added by Multidrop. 13. Plate incubated at room temperature in the dark for 30 minutes 14. Plate read in FUSION TABLE 2

This table demonstrates the inhibition of the growth of several cell lines with B-Raf mutations. The proportion of inhibition (as a percentage of the number of cells that do not incorporate BrdU) is shown in the last column TABLE 3

This table shows the inhibition of cell growth in cells treated with the MEK U0126 inhibitor TABLE 4 Inhibition of cell growth by BAY 43-9006.

Cell lines were incubated in the presence of increased levels of BAY 43-9006 and cell growth levels were determined by staining with sulforodamine B. IC50 values were determined by nonlinear regression analysis and are indicated. TABLE 5 Cell cycle analysis

WM-266.4 cells were treated with U0126, DMSO, siRNA for BRAF, siRNA for CRAF or the competitive siRNA control. The cells were incubated for 96 hours and the cells were fixed and stained with propridium iodide for cell cycle analysis by FACS. The proportion of cells in each phase of the cell cycle is demonstrated.

Computational aspects of detection

[00240] The detection of mutant B-Raf polypeptides and / or mutant B-raf nucleic acids can be automated to give a parallel and rapid compact selection of sample populations. Computerized methods for detecting the mutation are known in the art, and will generally involve the combination of a sequencing device, or another device capable of detecting sequence variation in polypeptides or nucleic acids, a data processing unit and an output device capable of to present the result in a form interpretable by a technician or physicist.

[00241] In a preferred aspect, therefore, the invention provides an automated method for detecting a mutation at a position of the target sequence in a nucleic acid derived from a naturally occurring primary human tumor encoding a B-Raf polypeptide comprising: sequencing a a naturally occurring primary human tumor nucleic acid amplification product sample to give a sample data set specifying a series of base pair identification data measured in a target domain that extends from one position to the initial sequence for a position of the final sequence, determine the presence or absence of the mutation in the sample with the proviso that the base pair identification data measured for the target sequence position corresponds with the reference base pair data for the target sequence position, and generate an output indicating the presence or absence of the mutation in the sample as indicated by the determining step.

[00242] Methods for sequencing and detecting sequential mutations are given above and are generally known in the art. The invention makes use of such methods in providing an apparatus for carrying out the process of the invention, an apparatus comprising: an operable sequence reading device for determining the sequence of a sample of a nucleic acid to give a set of sample data specifying base pair identification data measured in a target domain that extends from a position in the initial sequence to a position in the final sequence and a data analysis unit to receive the sample data set from the sequencing device and operable for determine the presence or absence of the mutation in the sample with the proviso that the identification data of the measured base pair for the position of the target sequence, correspond with the data of reference base pairs for the position of the target sequence.

[00243] Suitable sequence reading devices include automated sequencers, RFLP-analyzers and mobility deviation analysis devices. Advantageously the sequence of a target nucleic acid amplification product is analyzed, and the apparatus furthermore includes an amplification device such as a PCR (polymerase chain reaction) machine

[00244] Preferably, the apparatus also comprises an output device operable to generate an output indicating the presence or absence of the mutation in the sample determined by the data analysis unit. For example, the output device may comprise at least one of: a graphical user interface, an audible user interface, a printer, a storage medium for computer reading, and a computer interpretation medium and vehicle.

[00245] The invention can, moreover, be configured to detect the B-Raf protein itself. Thus, in a later aspect, the invention relates to an automated method for detecting a single mutation of amino acids in a B-Raf polypeptide from a naturally occurring primary human tumor, comprising: applying a marker to one or more target amino acids in a sample B-Raf polypeptide, read the sample after applying the marker to determine the presence or absence of the marker in the sample, to thereby indicate the presence or absence of the simple amino acid mutation in the sample, and generate an output indicating the presence or absence of the mutation of single amino acid in the sample as determined by the reading step.

[00246] The marker preferably comprises a linker that differentially binds to a wild-type B-Raf polypeptide without mutation of simple amino acids and to a mutant B-Raf polypeptide with the mutation. Preferential binding for any forms of B-Raf is possible in the context of the invention.

[00247] The invention furthermore provides an apparatus for detecting an amino acid mutation in a B-Raf polypeptide comprising: a marker protein protein loaded with a marker and operable to apply a marker to one or more target amino acids in a sample of the B polypeptide -Raf, and an operable marker reading device to determine the presence or absence of the marker in the sample, thus indicating the presence or absence of the single amino acid mutation in the sample.

[00248] The marker used can be an antibody, and the protein marker device can be configured to implement in an ELISA process.

[00249] Advantageously, the protein marker device comprises a microdevice which is preferably configured to read the sample optically.

[00250] Preferably, the apparatus comprises an operable output device for generating an output indicating the presence or absence of the simple amino acid mutation in the sample as determined by the marker reading device. Suitable output devices comprise at least one of: a graphical user interface, an audible user interface, a printer, a storage medium for reading by computer, and a means of vehicle for computer interpretation.

Uses of the Invention

[00251] The present invention provides new mutants of B-Raf polypeptides which are useful in detecting neoplastic conditions, and determining prognosis for individuals suffering from such conditions. In general, the presence of a mutation in B-Raf as described herein is associated with the presence of neoplasia.

[00252] In one aspect, the present invention provides a method for identifying cancer cells or tissue (such as malignant melanoma, colorectal cancer, breast cancer or NSCVLC) or identifiable cells or tissue that are predisposed to develop a neoplastic phenotype , comprising: amplifying at least part of a cell or tissue B-raf gene, analyzing the amplification product to detect a mutation in the B-raf gene as described herein, where a cell or tissue with one or more B- mutations raf is categorized as cancerous or being at an increased risk of developing a cancerous condition. Suitable amplification media include PCR and cloning.

[00253] In another embodiment, the present invention relates to a method for determining a prognosis in an individual suffering from cancer (such as malignant melanoma, colorectal cancer, breast cancer or NSCLC). The method comprises: amplifying the B-raf gene region as described above, analyzing the amplification products for evidence of the mutation as described above, and classifying an individual without mutations in the B-raf gene as being less likely to experience a relapse of the disease after therapy and / or surgery or with an increased chance of survival than a patient with one or more mutations in the region.

[00254] The techniques of the invention can also be employed to determine the course of therapy to which an individual should be exposed, based on the prognosis as stated above, an individual with a poor prognosis, is advantageously manipulated using a more aggressive therapy than an individual with a good prognosis.

[00255] The techniques according to the invention can be automated as necessary, for rapid examination of samples in the identification of potentially cancerous conditions. Generally, an automated process will comprise automated nucleic acid amplification of tissue or cell samples, detection of mutations in amplified nucleic acid, such as fluorescent detection, and / or revealing the presence of mutations. Exemplary automated modalities are described above.

[00256] The identification of the B-RAf mutant according to the invention can thus be used for diagnostic purposes to detect diagnose or monitor diseases, disorders, and / or conditions associated with the expression of the B-Raf mutant. In particular, the invention relates to the detection, diagnosis and / or monitoring of cancers associated with mutant B-Raf as described herein.

[00257] The invention provides a diagnostic assay for diagnosing cancer, comprising (a) assaying the expression of B-Raf mutant in an individual's cells or body fluid using one or more antibodies specific for the B-Raf mutant as indicated herein. The presence of the mutant B-raf transcript in an individual's biopsy tissue may indicate a predisposition for the development of the disease, or may provide a means to detect the disease before the actual clinical symptoms appear. A more definitive diagnosis of this type allows healthcare professionals to employ preventive measures or aggressive treatment early on, thus preventing the development of cancer progress.

[00258] Antibodies of the invention can be used for assaying protein levels in a biological sample using classical immunohistological methods known to those skilled in the art (for example, see Jalkane, et al., (1985) J. Cell. Biol. 101 : 976-985; Jalkanem et al., (1987) J. Cell Biol. 105: 3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase, radioisotopes, such as iodine (I125, I121), carbon (C14, sulfur (S35), tritium (H3), indium (In112) and technetium (TC99); luminescent labels such as luminol and fluorescent labels such as fluorescein and rhodamine and biotin.

[00259] In addition, mutations in B-raf can be detected by nucleic acid analysis as indicated herein. For example, the presence of mutations can be detected by sequencing or by SCCP analysis.

[00260] The present invention furthermore provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide, comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody that does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (for example, the antibody can be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a compound radioactive or a luminescent compound, or a second antibody, which recognizes the first antibody can be conjugated to a detectable substrate).

[00261] In another specific embodiment of the present invention, lit is a diagnostic kit for use in research serum containing specific antibodies to mutant B-Raf polypeptides as described herein. Such a kit can include a control antibody that does not react with the mutant B-RAf polypeptide. Such a kit can include a substantially isolated polypeptide antigen comprising an epitope that is specifically immunoreactive with at least one anti-B-Raf antibody. In addition, such a kit includes means for detecting the binding of the antibody to the antigen (e.g., the antibody can be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The kit's polypeptide antigen can also be attached to a solid support.

[00262] In an additional embodiment, the invention includes a diagnostic kit for use in the detection of serum containing antigens of the mutant B-RAf polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody immunoreactive with polypeptide or polynucleotide antigens and means for detecting the binding of the polynucleotide antigen or polypeptide to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody can be a monoclonal antibody. The kit detection means can include a second labeled monoclonal antibody. Alternatively, or in addition, the detection means may include a labeled competition antigen.

[00263] All publications mentioned in the above report are now incorporated by reference. Various modifications and variations of the described methods and system of the invention will become apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed, should not be unduly limited to such specific embodiments. In fact, several modifications of the modes described for carrying out the invention, evident to those skilled in molecular biology or related fields, are intended to be included in the scope of the following claims.

Claims (25)

1. Method for the detection of human oncogenic mutations, CHARACTERIZED by the fact that it comprises the steps of: (a) providing a sample of naturally occurring cellular material obtained from a human individual; (b) examining the nucleic acid material of at least part of one or more B-raf genes in said cell material; and (c) determining whether such a nucleic acid material comprises one or more point mutations in a sequence encoding a B-Raf polypeptide, wherein said point mutation occurs at one or more of positions 13881394, 1403, 1753, 1782, 1783, 1796, 1797, 1787 and 1786 of B-raf having the sequence in SEQ ID NO: 2. 2. Method for detecting human oncogenic mutations, CHARACTERIZED by the fact that it comprises the steps of: (a) providing a first sample of cellular material obtained from a naturally occurring tissue from an individual suspected of being cancerous, and a second sample of cellular material from a non-cancerous tissue from the same individual; (b) examining the nucleic acid material from at least part of one or more B-raf genes in both said samples of cellular material; and (c) determining whether such a nucleic acid material comprises one or more point mutations in a sequence encoding a B-RAF polypeptide; and said mutation being present in the naturally occurring cellular material of the tissue suspected of being cancerous, but not present in the cellular material of the non-cancerous tissue, wherein said dot mutation occurs in one or more of the positions 1388, 1394, 1403, 1753, 1782, 1783, 1796, 1797, 1787 and 1786 of B-raf having the sequence in SEQ ID NO: 2. 3. Method according to claim 1 or 2, CHARACTERIZED by the fact that said point mutation is G1388T, G1783C, TG1796-97AT, G1394T, G1394A, G1394C, G1403C, G1403A, G1753A, T1782G, G1388A, T1796A, T1787G or C1786G in B-raf which has the sequence in SEQ ID NO: 2. 4. Method for the detection of human oncogenic mutations, CHARACTERIZED by the fact that it comprises the steps of: (a) obtaining a sample of cellular material from an individual; (b) screening (screening) said sample with a linker that selectively binds to a mutant B-Raf polypeptide epitope, wherein the mutant B-Raf polypeptide has an amino acid mutation in one or more of the 463, 465 positions, 468, 585, 594, 595, 596 and 599 in B-Raf having the sequence in SEQ ID NO: 1; and (c) detecting one or more mutant B-Raf polypeptides that have an amino acid mutation at one or more of positions 463, 465, 468, 585, 594, 595, 596 and 599 in B-Raf having the sequence in SEQ ID NO: 1, in said sample. 5. Method according to claim 4, CHARACTERIZED by the fact that the mutant B-Raf polypeptide has an amino acid mutation selected from the group consisting of V599E, V599D, G595R, G465V, G465E, G465A, G468A, G468E , E585K, F594L, G595R, L596V, L596R and G463E. 6. Method according to claim 4 or 5, CHARACTERIZED by the fact that the ligand is an immunoglobulin. 7. Method, according to claim 6, CHARACTERIZED by the fact that the immunoglobulin is an antibody or an antigen-binding fragment thereof. 8. Automated method to detect a human mutation at a position of the target sequence in a nucleic acid encoding a B-Raf polypeptide, CHARACTERIZED by the fact that it comprises: sequencing a sample of a nucleic acid amplification product, to provide a set sample data specifying a series of base pair identification data measured in a target domain that extends from a position of the starting sequence to a position of the ending sequence; determining the presence or absence of the mutation in the sample with the proviso that the measured base pair identification data for the target sequence position match the reference base pair data for the target sequence position; and generate an output indicating the presence or absence of the mutation in the sample as established by the step of determining; wherein the nucleic acid comprises one or more point mutations; and wherein said point mutation occurs at one or more of positions 1388, 1394, 1403, 1753, 1782, 1783, 1796, 1797, 1787 and 1786 of B-raf having the sequence in SEQ ID NO: 2. 9. Automated method to detect a single human amino acid mutation in a B-Raf polypeptide, CHARACTERIZED by the fact that it comprises: applying a marker to one or more target amino acids in a sample of the B-Raf polypeptide; read the sample after application of the marker to determine the presence or absence of the marker in the sample, to thereby indicate the presence or absence of the single amino acid mutation in the sample; and generate an output indicating the presence or absence of the single amino acid mutation in the sample as determined by the reading step; wherein the B-Raf polypeptide comprises one or more mutations at positions 463, 465, 468, 585, 594, 595, 596 and 599 in B-Raf having the sequence in SEQ ID NO: 1. 10. Method according to claim 8, CHARACTERIZED by the fact that the marker comprises a linker that binds preferentially to a B-Raf polypeptide carrying the single amino acid mutation. 11. Method according to claim 8, CHARACTERIZED by the fact that the marker comprises a linker that binds preferentially to a wild-type B-Raf polypeptide without the single amino acid mutation. 12. Method, according to claim 8, CHARACTERIZED by the fact that the marker is an antibody. 13. Method, according to claim 8, CHARACTERIZED by the fact that it comprises an ELISA process. 14. Method, according to claim 8, CHARACTERIZED by the fact that the marker is applied using a microarrayer. 15. Method, according to claim 8, CHARACTERIZED by the fact that the sample is read optically. 16. Method, according to claim 8, CHARACTERIZED by the fact that the output is generated using a device that comprises at least one of: a graphical user interface; an audible user interface; a printer; a computer-readable storage medium; and a computer-interpretable carrier medium. 17. Method for identifying one or more compounds having anti-proliferative activity, CHARACTERIZED by the fact that it comprises the steps of: (a) providing one or more mutant B-Raf polypeptides, in which the one or more mutant B-Raf polypeptides have an amino acid mutation at one or more of positions 463, 465, 468, 585, 594, 595, 596 and 599 in B-Raf having the sequence in SEQ ID NO: 1; (b) contacting said polypeptide (s) with one or more compounds to be tested; and (c) detecting an interaction between said one or more compounds and said mutant polypeptides. 18. Method according to claim 17, CHARACTERIZED by the fact that the one or more mutant B-Raf polypeptides have an amino acid mutation selected from the group consisting of V599E, V599D, G595R, G465V, G465E, G465A, G468A, G468E, E585K, F594L, G595R, L596V, L596R and G463E. 19. Method, according to claim 17 or 18, CHARACTERIZED by the fact that the interaction is a binding interaction. 20. Assay for identification of one or more compounds having anti-proliferative activity, CHARACTERIZED by the fact that it comprises the steps of: (a) providing one or more mutant B-Raf polypeptides, in which the one or more mutant B-Raf polypeptides have an amino acid mutation at one or more of positions 463, 465, 468, 585, 594, 595, 596 and 599 in B-Raf having the sequence in SEQ ID NO: 1; (b) providing a downstream substrate for the B-Raf polypeptide; (c) detecting the modification of the substrate in the presence of the compound (s) to be tested. 21. Assay according to claim 20, CHARACTERIZED by the fact that the one or more mutant B-Raf polypeptides have an amino acid mutation selected from the group consisting of V599E, V599D, G595R, G465V, G465E, G465A, G468A, G468E, E585K, F594L, G595R, L596V, L596R and G463E. 22. Assay according to claim 20 or 21, CHARACTERIZED by the fact that the substrate modification is detected directly. 23. Assay according to claim 22, CHARACTERIZED by the fact that the substrate is an enzyme that modifies a second substrate, the second modification of which is detectable. 24. Assay according to claim 23, CHARACTERIZED by the fact that the substrate is MEK and the second substrate is MAPK. 25. Method or test, according to any one of claims 9 to 24, CHARACTERIZED by the fact that a reference level is determined for the test in the absence of the compound or compounds to be tested.

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